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The preliminary research on the risk factors of the Ib level lymphatic nodes metastasis in recurrent nasopharyngeal carcinoma
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Abstracts / Cell Biology International 32 (2008) S1eS67
INDUCTION OF DIFFERENTIATION AND APOPTOSIS IN MICE HEPATOCARCINOMA H22 CELLS INDUCED BY MICE EMBRYO EXTRACTIVE Ze Hua Yang 1, Ya Lan Cui 2, Xiao Bing Pan, Wei Hang Zheng, Shi Yun Cao 2 1 Department of Physiology, Yiyang Medical College, Yiyang, China 2 Department of Physiology, Medical College of Nanhua University, Hengyang, China Differentiation induction is a therapy for cancer. Searching for non-toxic and natural origin substances that induce the differentiation of cancer cells is a key for anticancer therapy. We investigated the effect of mice embryo extractive (E) on proliferation, differentiation, and apoptosis of H22 cells. In order to observe morphologic change of H22 cells, Giemsa stain was used. The nuclearcytoplasmic ratio and nuclear size of H22 cells were declined after treated with 2.5 % E contrast with control group and apoptosis cells were found. The result of MTT experiment showed that extractive (E) could effectively inhibit the proliferation of H22 cells in a dose-dependent and time-dependent manner. The activity of g-GT decreased (P<0.05) and the activity of ALP increased (P<0.05) in the cells treated with 2.5 % E after 120 hours compared with control group, indicating that the H22 cells differentiation degree was higher than control group’s. FCM analysis showed that apoptosis rate of E group was higher than control group’s (P<0.01).
EFFECTIVE ANTI-TUMOR RESPONSES ON NUDE MICE MODEL OF BREAST CANCER BY IMMUNOTHERAPY USING BREAST TUMOR-LYSATE PULSED DENDRITIC CELLS Wei Liu, Hui Jun Yi, Xu Dai, Xin Liu, Nan Wu, Dan Xu, Guang Xiu Lu Institute of Human Reproduction and Stem Cell Enginneering, Central South University, Changsha, Hunan, 410078, P.R.China Dendritic cells (DCs) loaded with breast tumor-lysate were injected into Balb/ c female nude mice inoculated with MCF-7 breast tumor cells in order to investigate the effects of DCs’ immunotherapy. A nude mice model of human breast cancer had been established by inoculating MCF-7 tumor cells in situ. Mononuclear cells (MNCs) were separated from health heparinized cord blood by Hyapque density gradient centrifugation, and MNCs were cultured in vitro by using RPMI1640 medium supplemented with GM-CSF, IL-4 and TNF-a. DCs were harvested at day 12, and cell counting and flow cytometry analysis were carried out. The capacity of DCs was assessed by the mixed leukocyte reaction (MLR). 65 tumor bearing nude mice models were randomized into 5 groups. Operations were carried out in group 1 to 4 to remove the solid tumors. Group 1 received DCs and CBMNCs, group 2 received CBMNCs, group 3 received DCs, group 4 received PBS, and group 5 received PBS only without operation. Tumor size was assessed by measuring the long (a) and short diameters (b) of tumors using calipers every three days and the tumor volume was calculated by 1/6mab2. Totally 4 mice died of operation in group 3 and 4. After immunotherapy for 3 months, a high survival rate was shown in group 1 (93.33 %) which was injected with DCs and CBMNCs and it was statistically significant when compared to group 4 (60%) which was injected with PBS only (P¼0.026, by a Chi-square test). In contrast, the tumor recurrence rate of group 1 (40%) was markedly lower than group 4 (77.8%) and group 3 (87.5%), and was statistically significant compared to group 4 (P¼0.027,by a Chi-square test) and group 3 (P¼0.029, by a Chisquare test). One-way ANOVA about the tumor volumes among these groups showed statistic significance (P¼ 0.004, by least-significant difference test) between group1 (516.66 mm3) and group4 (5052.85mm3). The tumor volume of group 5 (10285.73 mm3) was the biggest and data showed statistic significance (P<0.05, by least-significant difference test) compared to other four groups. In conclusion, our results show that vaccination of DCs pulsed with tumor lysates could induce anti-tumor responses in nude mice model of breast cancer.
THE PRELIMINARY RESEARCH ON THE RISK FACTORS OF THE IB LEVEL LYMPHATIC NODES METASTASIS IN RECURRENT NASOPHARYNGEAL CARCINOMA Wei Yi 1, Jin Tian Li 1,2, Yun Fei Xia 1,2 1 Cancer Center, Sun Yat-Sen University, Guangzhou, China 2 State key laboratory of oncology in Southern China, Guangzhou, Guangdong Province, China To find some predictable risk index for Ib level lymphatic nodes metastasis in recurrent NPC (nasopharyngeal carcinoma), 51 patients with pathologically confirmed recurrent NPC treated in the cancer center of Sun Yat-Sen University from 1995 to 1998 were studied. We selected factors as the local invasion and the expression levels of VEGF, P53, MDM2, P21WAF1, P21RAS proteins were analyzed. There were 9 cases with Ib level lymphatic nodes metastasis. The Ib level lymphatic nodes metastasis rates had no significant difference between the different sex, T stages, N stages and TNM stages. The Ib level lymphatic nodes metastasis rate was 30.4 % (7/23) in the group with the nasal cavity invasion, only 7.1 % (2/28) in the group without nasal cavity invasion, but the difference was not significant in statistics (P¼0.061). When we divided the patients into 2 groups of high and low expression level of VEGF protein, or P53, MDM2, P21WAF1, P21RAS protein, the Ib level lymphatic nodes metastasis rates between high and low expression groups had no significant difference. When the NPC invaded the nasal cavity coordinated with high expression level of VEGF, the Ib level lymphatic nodes metastasis rate (37.5 %) (6/16) was significantly higher than others (8.6 %)(3/35) (P<0.05). When we used other index like P53, MDM2, P21RAS, or P21WAF1 protein to replace VEGF, the difference of Ib level lymphatic nodes metastasis rate were not significant statistically. We concluded when the NPC invaded the nasal cavity coordinated with high level expression of VEGF, the Ib level lymphatic nodes metastasis rate rose significantly in recurrent NPC.
HYPOXIA INDUCIBLE FACTOR-1 INFLUENCES SENSITIVITY TO PACLITAXEL OF HUMAN LUNG CANCER CELL LINES UNDER NORMOXIC CONDITIONS Li Hua Zeng 1, Guo Zhen Guo 1, Shinae Kizaka-Kondoh 2, Satoshi Itasaka 2, Masahiro Hiraoka 2 1 Department of Radiation Medicine, Fourth Military Medical University, 17 Chargle West Road, Xi’an, Shaanxi 710032, China 2 Department of Radiation Oncology and Image-applied Therapy, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Paclitaxel (PTX) is an anticancer drug that is effective against a wide range of solid tumors. The effect of PTX on two human lung cancer cell lines, PC14PE6 and NCI-H441 cells, was examined in an orthotopically transplanted animal model with an in vivo imaging devise. Although PTX effectively suppressed tumor growth and improved survival rate in NCI-H441, it did not influence these in PC14PE6. In vitro experiments confirmed that PC14PE6 cells were resistant to PTX under normoxic conditions and that both cell lines were resistant to PTX under hypoxic conditions. It was found that the expression level of endogenous hypoxia inducible factor (HIF)-1a in PC14PE6 was much higher than that in NCI-H441 cells under normoxic conditions. Furthermore, sensitivity to PTX in these cell lines was reversed when HIF-1a expression was decreased by siRNA specific to HIF-1a in PC14PE6 and increased by overexpression of the exogenous HIF-1a gene in NCI-H441. These results suggest that HIF-1 influenced the PTX sensitivity of these cells. The authors further examined b-tubulin, a target molecule of PTX, with western blotting and immunohistochemical analysis in these cells. The expression level of b-tubulin was comparable in these cells under both normoxic and hypoxic conditions while the distribution of b-tubulin and cell morphology were changed according to HIF-1a expression levels, suggesting that HIF-1 influenced the conformation and dynamics of microtubules. These data support the potential development of HIF-1 targeted approaches in combination with PTX, where drug resistance tends to contribute to treatment failure.
Abstracts / Cell Biology International 32 (2008) S1eS67
INDUCTION OF DIFFERENTIATION AND APOPTOSIS IN MICE HEPATOCARCINOMA H22 CELLS INDUCED BY MICE EMBRYO EXTRACTIVE Ze Hua Yang 1, Ya Lan Cui 2, Xiao Bing Pan, Wei Hang Zheng, Shi Yun Cao 2 1 Department of Physiology, Yiyang Medical College, Yiyang, China 2 Department of Physiology, Medical College of Nanhua University, Hengyang, China Differentiation induction is a therapy for cancer. Searching for non-toxic and natural origin substances that induce the differentiation of cancer cells is a key for anticancer therapy. We investigated the effect of mice embryo extractive (E) on proliferation, differentiation, and apoptosis of H22 cells. In order to observe morphologic change of H22 cells, Giemsa stain was used. The nuclearcytoplasmic ratio and nuclear size of H22 cells were declined after treated with 2.5 % E contrast with control group and apoptosis cells were found. The result of MTT experiment showed that extractive (E) could effectively inhibit the proliferation of H22 cells in a dose-dependent and time-dependent manner. The activity of g-GT decreased (P<0.05) and the activity of ALP increased (P<0.05) in the cells treated with 2.5 % E after 120 hours compared with control group, indicating that the H22 cells differentiation degree was higher than control group’s. FCM analysis showed that apoptosis rate of E group was higher than control group’s (P<0.01).
EFFECTIVE ANTI-TUMOR RESPONSES ON NUDE MICE MODEL OF BREAST CANCER BY IMMUNOTHERAPY USING BREAST TUMOR-LYSATE PULSED DENDRITIC CELLS Wei Liu, Hui Jun Yi, Xu Dai, Xin Liu, Nan Wu, Dan Xu, Guang Xiu Lu Institute of Human Reproduction and Stem Cell Enginneering, Central South University, Changsha, Hunan, 410078, P.R.China Dendritic cells (DCs) loaded with breast tumor-lysate were injected into Balb/ c female nude mice inoculated with MCF-7 breast tumor cells in order to investigate the effects of DCs’ immunotherapy. A nude mice model of human breast cancer had been established by inoculating MCF-7 tumor cells in situ. Mononuclear cells (MNCs) were separated from health heparinized cord blood by Hyapque density gradient centrifugation, and MNCs were cultured in vitro by using RPMI1640 medium supplemented with GM-CSF, IL-4 and TNF-a. DCs were harvested at day 12, and cell counting and flow cytometry analysis were carried out. The capacity of DCs was assessed by the mixed leukocyte reaction (MLR). 65 tumor bearing nude mice models were randomized into 5 groups. Operations were carried out in group 1 to 4 to remove the solid tumors. Group 1 received DCs and CBMNCs, group 2 received CBMNCs, group 3 received DCs, group 4 received PBS, and group 5 received PBS only without operation. Tumor size was assessed by measuring the long (a) and short diameters (b) of tumors using calipers every three days and the tumor volume was calculated by 1/6mab2. Totally 4 mice died of operation in group 3 and 4. After immunotherapy for 3 months, a high survival rate was shown in group 1 (93.33 %) which was injected with DCs and CBMNCs and it was statistically significant when compared to group 4 (60%) which was injected with PBS only (P¼0.026, by a Chi-square test). In contrast, the tumor recurrence rate of group 1 (40%) was markedly lower than group 4 (77.8%) and group 3 (87.5%), and was statistically significant compared to group 4 (P¼0.027,by a Chi-square test) and group 3 (P¼0.029, by a Chisquare test). One-way ANOVA about the tumor volumes among these groups showed statistic significance (P¼ 0.004, by least-significant difference test) between group1 (516.66 mm3) and group4 (5052.85mm3). The tumor volume of group 5 (10285.73 mm3) was the biggest and data showed statistic significance (P<0.05, by least-significant difference test) compared to other four groups. In conclusion, our results show that vaccination of DCs pulsed with tumor lysates could induce anti-tumor responses in nude mice model of breast cancer.
THE PRELIMINARY RESEARCH ON THE RISK FACTORS OF THE IB LEVEL LYMPHATIC NODES METASTASIS IN RECURRENT NASOPHARYNGEAL CARCINOMA Wei Yi 1, Jin Tian Li 1,2, Yun Fei Xia 1,2 1 Cancer Center, Sun Yat-Sen University, Guangzhou, China 2 State key laboratory of oncology in Southern China, Guangzhou, Guangdong Province, China To find some predictable risk index for Ib level lymphatic nodes metastasis in recurrent NPC (nasopharyngeal carcinoma), 51 patients with pathologically confirmed recurrent NPC treated in the cancer center of Sun Yat-Sen University from 1995 to 1998 were studied. We selected factors as the local invasion and the expression levels of VEGF, P53, MDM2, P21WAF1, P21RAS proteins were analyzed. There were 9 cases with Ib level lymphatic nodes metastasis. The Ib level lymphatic nodes metastasis rates had no significant difference between the different sex, T stages, N stages and TNM stages. The Ib level lymphatic nodes metastasis rate was 30.4 % (7/23) in the group with the nasal cavity invasion, only 7.1 % (2/28) in the group without nasal cavity invasion, but the difference was not significant in statistics (P¼0.061). When we divided the patients into 2 groups of high and low expression level of VEGF protein, or P53, MDM2, P21WAF1, P21RAS protein, the Ib level lymphatic nodes metastasis rates between high and low expression groups had no significant difference. When the NPC invaded the nasal cavity coordinated with high expression level of VEGF, the Ib level lymphatic nodes metastasis rate (37.5 %) (6/16) was significantly higher than others (8.6 %)(3/35) (P<0.05). When we used other index like P53, MDM2, P21RAS, or P21WAF1 protein to replace VEGF, the difference of Ib level lymphatic nodes metastasis rate were not significant statistically. We concluded when the NPC invaded the nasal cavity coordinated with high level expression of VEGF, the Ib level lymphatic nodes metastasis rate rose significantly in recurrent NPC.
HYPOXIA INDUCIBLE FACTOR-1 INFLUENCES SENSITIVITY TO PACLITAXEL OF HUMAN LUNG CANCER CELL LINES UNDER NORMOXIC CONDITIONS Li Hua Zeng 1, Guo Zhen Guo 1, Shinae Kizaka-Kondoh 2, Satoshi Itasaka 2, Masahiro Hiraoka 2 1 Department of Radiation Medicine, Fourth Military Medical University, 17 Chargle West Road, Xi’an, Shaanxi 710032, China 2 Department of Radiation Oncology and Image-applied Therapy, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Paclitaxel (PTX) is an anticancer drug that is effective against a wide range of solid tumors. The effect of PTX on two human lung cancer cell lines, PC14PE6 and NCI-H441 cells, was examined in an orthotopically transplanted animal model with an in vivo imaging devise. Although PTX effectively suppressed tumor growth and improved survival rate in NCI-H441, it did not influence these in PC14PE6. In vitro experiments confirmed that PC14PE6 cells were resistant to PTX under normoxic conditions and that both cell lines were resistant to PTX under hypoxic conditions. It was found that the expression level of endogenous hypoxia inducible factor (HIF)-1a in PC14PE6 was much higher than that in NCI-H441 cells under normoxic conditions. Furthermore, sensitivity to PTX in these cell lines was reversed when HIF-1a expression was decreased by siRNA specific to HIF-1a in PC14PE6 and increased by overexpression of the exogenous HIF-1a gene in NCI-H441. These results suggest that HIF-1 influenced the PTX sensitivity of these cells. The authors further examined b-tubulin, a target molecule of PTX, with western blotting and immunohistochemical analysis in these cells. The expression level of b-tubulin was comparable in these cells under both normoxic and hypoxic conditions while the distribution of b-tubulin and cell morphology were changed according to HIF-1a expression levels, suggesting that HIF-1 influenced the conformation and dynamics of microtubules. These data support the potential development of HIF-1 targeted approaches in combination with PTX, where drug resistance tends to contribute to treatment failure.