Purpose: While secretagogue-induced diarrhea is rich in chloride (Cl 7 ) and bicarbonate (HCO 3 7... more Purpose: While secretagogue-induced diarrhea is rich in chloride (Cl 7 ) and bicarbonate (HCO 3 7 ) anions, little is known about diarrhea or its anionic composition following irradiation. We performed studies to characterize the differences between cyclic adenosine monophosphate (cAMP)-stimulated anion secretions in irradiated and non-irradiated mice. Materials and methods: HCO 3 7 secretion was examined in basal, cAMP-stimulated, and irradiated jejunal tissues from BALB/c (Bagg albino) mice. The abdomens of the mice were g-irradiated using a caesium-137 source. Results: Ussing-chamber experiments performed in an HCO 3 7 -containing, Cl 7 -free solution on the bath side showed inhibition of HCO 3 7 in irradiated mice. Non-irradiated mice exhibited bumetanide-sensitive and insensitive current, while irradiated mice displayed bumetanide-sensitive current. pH-stat experiments showed inhibition of basal and cAMPstimulated HCO 3 7 secretions in irradiated mice. Immunohistochemistry and Western blot analysis displayed a sodiumbicarbonate cotransporter expression in the villus and not the crypt of non-irradiated mice, while its expression and protein levels decreased in irradiated mice. Conclusions: Anion secretions in irradiated mice, being primarily Cl 7 and minimally HCO 3 7 , differ from that of secretagogue-induced anion secretions. Understanding anion loss will help us correct electrolyte imbalances, while reduced HCO 3 7 secretion in the upper-gastrointestinal tract might also have implications for irradiation-induced nausea and vomiting.
To study the mechanism how human sperm membrane-bound hyaluronidase (PH20) promotes the gowth of ... more To study the mechanism how human sperm membrane-bound hyaluronidase (PH20) promotes the gowth of human breast cancer. Full-length cDNA of human PH20 was transfected into human breast cancer cell line MDA231. The transfectant MDA231-PH20 was then implanted into the chorio-allantoic membrane (CAM) of chicken embryo to form a tumor. Four days after implantation, the tumors were resected to be weighed. The angiogenesis in tumor tissue was examined by immunohistochemistry. Trans-well cell culture was used to study the effect of MDA231-PH20 on the growth of adult bovine aortic endothelial cells (ABAE). The expression of fibroblast growth factor-2 (FGF-2) in the tumor cells was investigated by Western blotting. ELISA was used to examine the secretion of FGF-2 and hyaluronic acid. The same amount of empty vector pcDNA3, instead of PH20, was transfected into human breast cancer cell line MDA231 as control group. The average weight of tumor four days after implantation was 44.7 mg +/- 10.2 mg in the MDA231-PH20 group, and was 21.3 mg +/- 2.8 mg in the control group (t = 2.418, P = 0.038). Neogenetic vessels increased remarkably in MDA231-PH20 tumor tissues. The expression of FGF-2 protein was much higher in MDA231-PH20 cells. The FGF content and HA secretion were higher in the MDA231-PH20 group than in the control group (8.10 pg/ml +/- 1.56 pg/ml vs. 3.94 pg/ml +/- 0.82 pg/ml, and 1 220 ng/ml +/- 254 ng/ml vs. 462 ng/ml +/- 96 ng/ml, all P < 0.01). The growth of ABAE cells was significantly accelerated after co-culture with MDA231-PH20 transfectant. PH20 may promote the growth of human breast cancer by accelerating the release of FGF-2 from tumor cells, decomposing HA into small fragments, and promoting angiogenesis.
Hyaluronan synthase 3 (HAS3) is responsible for the production of both secreted and cell-associat... more Hyaluronan synthase 3 (HAS3) is responsible for the production of both secreted and cell-associated forms of hyaluronan and is the most active of the three isoforms of this enzyme in adults. In this study, the cDNA for human HAS3 was cloned and characterized. The open reading frame consisted of 1659 bp coding for 553 amino acids with a deduced molecular weight of about 63,000 and isoelectric pH of 8.70. The sequence of human HAS3 displayed a 53% identity to HAS1 and a 67% identity to HAS2. It also contained a signal peptide and six potential transmembrane domains, suggesting that it was associated with the plasma membrane. To evaluate the physiological role of human HAS3, expression vectors for this protein were transfected into TSU cells (a prostate cancer cell line), and the phenotypic changes in these cells were examined. The enhanced expression of hyaluronan in the transfected cells was demonstrated by dot blot analysis and ELISA. These cells were found to differ from their vectortransfected counterparts with respect to the following: (a) they grew at a faster rate in high (but not low) density cultures;
Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfecte... more Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfected with fibroblast growth factor (FGF) 1 have frequent micrometastases, but macrometastases are not observed. i.v. injection of FGF-1-transfected tumor cells produced no pulmonary macrometastases, and removal of primary tumors resulted in the disappearance of spontaneous micrometastases. Thus, failure of micrometastases to proliferate was not due to inhibitory factors released from the primary tumor, and the presence of the primary tumor is required for maintenance of the micrometastases. This indicates that the micrometastases result from continued seeding from the primary tumor balanced by clearance from the metastatic site. Tumor emboli trapped in the vessels of lungs and lymph nodes and single tumor cells observed in the pulmonary vein implied that FGF-1-overexpressing MCF-7 cells are deficient in their ability to extravasate. The frequency of tumor cells incorporating bromodeoxyuridi...
Advances in experimental medicine and biology, 2014
The tumor vascular system, which is critical to the survival and growth of solid tumors, has been... more The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 μM, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellul...
ORL; journal for oto-rhino-laryngology and its related specialties, 2008
We studied the relationships between the growth, metastasis and angiogenesis of laryngeal carcino... more We studied the relationships between the growth, metastasis and angiogenesis of laryngeal carcinoma and extracellular matrix protein-1 (ECM1) and hyaluronan (HA). Thirty-three cases of benign and malignant laryngeal tumors were included. Using immunohistochemical staining, we examined the expression of ECM1 and HA in tumors and metastatic lymph nodes and correlated it with counts of microvessel density. The expression of ECM1 (p = 0.004) and HA (p = 0.036) was significantly different between benign and malignant tumors. The expression of ECM1 was strongly associated with microvessel density (Spearman's rho = 0.513, p = 0.017), with the strongest expression present within caner nests. With respect to HA, expression was significantly associated with more advanced clinical TNM stage (Spearman's rho = 0.521, p = 0.015). The link was stronger in cases where metastasis or recurrence occurred than in those without complication (p = 0.014). No significant relationship was found betw...
Advances in experimental medicine and biology, 2007
Triptolide (TPL), a small molecule purified from the herb Tripterygium wilfordii, has potential c... more Triptolide (TPL), a small molecule purified from the herb Tripterygium wilfordii, has potential clinical application for suppression of chronic autoimmune disorders and inhibition of tumor growth. However, its mechanism of action is largely unknown. In this study, the effect of TPL on mitochondria was explored with a panel of molecular probes that detect the alteration of mitochondrial functions. When Lewis lung carcinoma (LLC) cells were treated with different doses of TPL for four hours, impaired mitochondrial functions were detected. This included an increased production of reactive oxygen species, the opening of the transition pore of mitochondria, the depolarization of the mitochondria membrane, the inhibition of the production of ATP and increased release of ATP as well as the induction of apoptosis. It is likely that by impairment of mitochondrial function, TPL exerts its inhibitory effect on growth of tumor and progression of inflammatory disease.
In this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine... more In this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine cartilage by affinity chromatography and found to have both antitumorigenic and antiangiogenic properties. Metastatin was able to block the formation of tumor nodules in the lungs of mice inoculated with B16BL6 melanoma or Lewis lung carcinoma cells. Single i.v. administration of Metastatin into chicken embryos inhibited the growth of both B16BL6 mouse melanoma and TSU human prostate cancer cells growing on the chorioallantoic membrane. The in vivo biological effect may be attributed to the antiangiogenic activity because Metastatin is able to inhibit the migration and proliferation of cultured endothelial cells as well as vascular endothelial growth factor-induced angiogenesis on the chorioallantoic membrane. In each case, the effect could be blocked by either heat denaturing the Metastatin or premixing it with hyaluronan, suggesting that its activity critically depends on its ability ...
Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfecte... more Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfected with fibroblast growth factor (FGF) 1 have frequent micrometastases, but macrometastases are not observed. i.v. injection of FGF-1-transfected tumor cells produced no pulmonary macrometastases, and removal of primary tumors resulted in the disappearance of spontaneous micrometastases. Thus, failure of micrometastases to proliferate was not due to inhibitory factors released from the primary tumor, and the presence of the primary tumor is required for maintenance of the micrometastases. This indicates that the micrometastases result from continued seeding from the primary tumor balanced by clearance from the metastatic site. Tumor emboli trapped in the vessels of lungs and lymph nodes and single tumor cells observed in the pulmonary vein implied that FGF-1-overexpressing MCF-7 cells are deficient in their ability to extravasate. The frequency of tumor cells incorporating bromodeoxyuridi...
A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediat... more A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediated motility (RHAMM), and metastatin inhibit tumor growth and metastasis. To determine whether the HA binding motif is the element responsible for the antitumor effect of this family of proteins, we examined the biological activity of a 42-amino acid peptide (designated as BH-P) that contains three HA binding motifs [B(X(7))B] from human brain HA binding protein. In initial experiments with cultured cells, we found that synthetic BH-P inhibited the proliferation and colony formation of tumor cells. It also blocked the growth of tumors on the chorioallantoic membranes of 10-day chicken embryos. In addition, MDA-435 melanoma cells that had been transfected with an expression vector for BH-P grew at a slower rate in nude mice than the vector-alone transfectants. Final studies revealed that the BH-P could activate caspase-8, caspase-3, and poly(ADP-ribose) polymerase and trigger the apoptosis...
Increased extracellular matrix 1 (ECM1) expression, as determined by real-time reverse transcript... more Increased extracellular matrix 1 (ECM1) expression, as determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), has been recently proposed as a novel and independent diagnostic marker for malignant thyroid nodules. However ECM1 protein expression has not been previously evaluated in thyroid tumors. Real-time RT-PCR is not widely available and this has important implications for the widespread use of the expression of this gene for diagnosis. The purpose of the current study is to evaluate and compare ECM1 expression by real-time RT-PCR with immunohistochemistry (IHC) as diagnostic aids in thyroid neoplasms. ECM1 expression was studied in normal thyroid tissues (n = 14) and primary thyroid tumors (19 benign, 30 malignant) using both techniques. The performance characteristics of ECM1 expression were examined with receiver operating characteristic curves and the resulting area under the curve. ECM1 was highly expressed in thyroid carcinomas, compared with benign thyroid tissues, both at the mRNA (P < 0.01) and protein (P = 0.06) levels. However, ECM1 mRNA expression appeared to be a more sensitive marker of thyroid malignancy than IHC (AUC = 0.77 and 0.65, respectively). ECM1 expression by both methods was useful in identifying malignant follicular, but not Hürthle cell neoplasms. We confirm that ECM1 expression has potential utility as an independent diagnostic marker for thyroid cancer, although, performance characteristics were lower than previously published. Furthermore, IHC was not as sensitive as real-time RT-PCR in identifying malignant lesions. Prospective studies are needed to further clarify the role of ECM1 expression as a diagnostic marker.
Advances in Experimental Medicine and Biology, 2003
Breast tumors expressing no detectable FGFs (MCF-7) were compared with tumors transfected with FG... more Breast tumors expressing no detectable FGFs (MCF-7) were compared with tumors transfected with FGF4 or FGF1 (FGF4/MCF-7 or FGF1/MCF-7), and with MDA-MB-435, which produce endogenous FGF2. Tumor blood flow was measured by 133Xe diffusion, oxygen distribution was measured by Eppendorf pO2 histography, and vascular density was measured by CD31 staining. Tumors that overexpress angiogenic factors grew at a rate far exceeding that of MCF-7. The FGF producing tumors also had much higher metastatic rates to lung. Tumor blood flow was significantly higher in the two FGF-transfected xenografts compared with the parent MCF7. Median tumor pO2 was also higher, and tumor oxygenation was preserved even for large tumors. The vascular density as determined by CD31 staining, however, was not markedly increased in tumors overexpressing angiogenic factors. We found that angiogenic factors preserve and augment neovascular function, thus facilitating tumor growth and progression.
Advances in experimental medicine and biology, 2013
Tumor hypoxia is probably the most important not yet measurable factor that predicts the outcome ... more Tumor hypoxia is probably the most important not yet measurable factor that predicts the outcome of cancer therapy. Hypoxic tumors are resistant to radiation, chemotherapy, and surgery. They signal tumor cells to grow, invade, survive cytotoxic-factor assault, and increase metastatic activity. Therapies aimed at reversing hypoxia-related treatment resistance or normalizing hypoxia are proven effective with level 1 evidence. The weak link remains the lack of satisfactory methods of measurement of tumor oxygenation.
Advances in Experimental Medicine and Biology, 2005
Hyaluronan (HA), a large negatively-charged polysaccharide, is a major component of vessel basal ... more Hyaluronan (HA), a large negatively-charged polysaccharide, is a major component of vessel basal membrane. HA is expressed by a variety of cells, including tumor and endothelial cells. We hypothesized that HA could be up-regulated by hypoxia to enhance vessel formation. To determine the effect of hypoxia on the production of HA, tumor cells were treated with either media alone (control) or a hypoxia inducer (CoCl or NaN3) for 24 h. The level of HA in the media was then measured by ELISA. The results showed that both CoCl and NaN3 induced the production of HA. Since the low molecular weight form of HA (SMW) possesses pro-angiogenic properties, we investigated whether hypoxia-induced HA can be processed into SMW. Under hypoxic conditions, the activity of hyaluronidase, the enzyme responsible for degrading HA, was measured by an ELISA-like assay. The activity of hyaluronidase was shown to be up-regulated by hypoxia and, further, could carry out the function of processing HA into SMW. In addition, the hypoxic areas of tumor tissues were stained strongly with biotinylated HA-binding proteins, indicating that the level of HA was high compared to the oxic areas. This study demonstrates that hypoxia can stimulate the production of HA and the activity of hyaluronidase, which may promote angiogenesis as a compensation mechanism for hypoxia.
Chick chorioallantoic membrane (CAM) angiogenesis assay has been widely used for finding drugs ta... more Chick chorioallantoic membrane (CAM) angiogenesis assay has been widely used for finding drugs targeting new blood vessel development in cancer research. In addition to the setup materials and protocols, laboratory findings depend on the quantification and analysis of microscopic blood vessel images. However, it is still a challenging problem because of the high complexity of blood vessel branching structures. We applied preprocessing on CAM microscopic images by keeping the integrity of minor branches in the vessel structure. We then proposed an efficient way to automatically extract blood vessel centerlines based on vector tracing starting from detected seed points. Finally, all branches were coded to construct an abstract model of the branching structure, which enabled more accurate modeling for in-depth analysis. The framework was applied in quantifying Icaritin (ICT) inhibition effects on angiogenesis in a CAM model. Experimental results showed the high accuracy in blood vessel...
Mitochondria play pivotal roles in cellular handling of oxygen and in apoptosis, the ordered suic... more Mitochondria play pivotal roles in cellular handling of oxygen and in apoptosis, the ordered suicide response of cells to irradiation. The involvement of expression products from the 16.5 kb human mitochondrial genome in these activities has been studied widely. However, little is known about effects of irradiation on mammalian mitochondrial DNA (mtDNA). The relative lack of mtDNA repair mechanisms compared with nuclear DNA (nDNA) predicts particular vulnerability to irradiation. Using a technique developed to ascertain mtDNA:nDNA ratios, we previously showed that this ratio increases dramatically in murine small bowel within 48 hours following whole body irradiation. We now report that those levels continue to rise for four days and remain elevated at close to that level beyond 30 days after 5 Gy of irradiation.We further demonstrate that levels of the mtDNA-specific DNA polymerase-γ (Pol-γ ) also show a sharp and sustained increase during this time course after a 2-Gy dose. Paradoxically, transcription factor A (TFAM), exhibited the directly opposite response.
Advances in experimental medicine and biology, 2009
The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondr... more The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclu...
Purpose: While secretagogue-induced diarrhea is rich in chloride (Cl 7 ) and bicarbonate (HCO 3 7... more Purpose: While secretagogue-induced diarrhea is rich in chloride (Cl 7 ) and bicarbonate (HCO 3 7 ) anions, little is known about diarrhea or its anionic composition following irradiation. We performed studies to characterize the differences between cyclic adenosine monophosphate (cAMP)-stimulated anion secretions in irradiated and non-irradiated mice. Materials and methods: HCO 3 7 secretion was examined in basal, cAMP-stimulated, and irradiated jejunal tissues from BALB/c (Bagg albino) mice. The abdomens of the mice were g-irradiated using a caesium-137 source. Results: Ussing-chamber experiments performed in an HCO 3 7 -containing, Cl 7 -free solution on the bath side showed inhibition of HCO 3 7 in irradiated mice. Non-irradiated mice exhibited bumetanide-sensitive and insensitive current, while irradiated mice displayed bumetanide-sensitive current. pH-stat experiments showed inhibition of basal and cAMPstimulated HCO 3 7 secretions in irradiated mice. Immunohistochemistry and Western blot analysis displayed a sodiumbicarbonate cotransporter expression in the villus and not the crypt of non-irradiated mice, while its expression and protein levels decreased in irradiated mice. Conclusions: Anion secretions in irradiated mice, being primarily Cl 7 and minimally HCO 3 7 , differ from that of secretagogue-induced anion secretions. Understanding anion loss will help us correct electrolyte imbalances, while reduced HCO 3 7 secretion in the upper-gastrointestinal tract might also have implications for irradiation-induced nausea and vomiting.
To study the mechanism how human sperm membrane-bound hyaluronidase (PH20) promotes the gowth of ... more To study the mechanism how human sperm membrane-bound hyaluronidase (PH20) promotes the gowth of human breast cancer. Full-length cDNA of human PH20 was transfected into human breast cancer cell line MDA231. The transfectant MDA231-PH20 was then implanted into the chorio-allantoic membrane (CAM) of chicken embryo to form a tumor. Four days after implantation, the tumors were resected to be weighed. The angiogenesis in tumor tissue was examined by immunohistochemistry. Trans-well cell culture was used to study the effect of MDA231-PH20 on the growth of adult bovine aortic endothelial cells (ABAE). The expression of fibroblast growth factor-2 (FGF-2) in the tumor cells was investigated by Western blotting. ELISA was used to examine the secretion of FGF-2 and hyaluronic acid. The same amount of empty vector pcDNA3, instead of PH20, was transfected into human breast cancer cell line MDA231 as control group. The average weight of tumor four days after implantation was 44.7 mg +/- 10.2 mg in the MDA231-PH20 group, and was 21.3 mg +/- 2.8 mg in the control group (t = 2.418, P = 0.038). Neogenetic vessels increased remarkably in MDA231-PH20 tumor tissues. The expression of FGF-2 protein was much higher in MDA231-PH20 cells. The FGF content and HA secretion were higher in the MDA231-PH20 group than in the control group (8.10 pg/ml +/- 1.56 pg/ml vs. 3.94 pg/ml +/- 0.82 pg/ml, and 1 220 ng/ml +/- 254 ng/ml vs. 462 ng/ml +/- 96 ng/ml, all P < 0.01). The growth of ABAE cells was significantly accelerated after co-culture with MDA231-PH20 transfectant. PH20 may promote the growth of human breast cancer by accelerating the release of FGF-2 from tumor cells, decomposing HA into small fragments, and promoting angiogenesis.
Hyaluronan synthase 3 (HAS3) is responsible for the production of both secreted and cell-associat... more Hyaluronan synthase 3 (HAS3) is responsible for the production of both secreted and cell-associated forms of hyaluronan and is the most active of the three isoforms of this enzyme in adults. In this study, the cDNA for human HAS3 was cloned and characterized. The open reading frame consisted of 1659 bp coding for 553 amino acids with a deduced molecular weight of about 63,000 and isoelectric pH of 8.70. The sequence of human HAS3 displayed a 53% identity to HAS1 and a 67% identity to HAS2. It also contained a signal peptide and six potential transmembrane domains, suggesting that it was associated with the plasma membrane. To evaluate the physiological role of human HAS3, expression vectors for this protein were transfected into TSU cells (a prostate cancer cell line), and the phenotypic changes in these cells were examined. The enhanced expression of hyaluronan in the transfected cells was demonstrated by dot blot analysis and ELISA. These cells were found to differ from their vectortransfected counterparts with respect to the following: (a) they grew at a faster rate in high (but not low) density cultures;
Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfecte... more Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfected with fibroblast growth factor (FGF) 1 have frequent micrometastases, but macrometastases are not observed. i.v. injection of FGF-1-transfected tumor cells produced no pulmonary macrometastases, and removal of primary tumors resulted in the disappearance of spontaneous micrometastases. Thus, failure of micrometastases to proliferate was not due to inhibitory factors released from the primary tumor, and the presence of the primary tumor is required for maintenance of the micrometastases. This indicates that the micrometastases result from continued seeding from the primary tumor balanced by clearance from the metastatic site. Tumor emboli trapped in the vessels of lungs and lymph nodes and single tumor cells observed in the pulmonary vein implied that FGF-1-overexpressing MCF-7 cells are deficient in their ability to extravasate. The frequency of tumor cells incorporating bromodeoxyuridi...
Advances in experimental medicine and biology, 2014
The tumor vascular system, which is critical to the survival and growth of solid tumors, has been... more The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 μM, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellul...
ORL; journal for oto-rhino-laryngology and its related specialties, 2008
We studied the relationships between the growth, metastasis and angiogenesis of laryngeal carcino... more We studied the relationships between the growth, metastasis and angiogenesis of laryngeal carcinoma and extracellular matrix protein-1 (ECM1) and hyaluronan (HA). Thirty-three cases of benign and malignant laryngeal tumors were included. Using immunohistochemical staining, we examined the expression of ECM1 and HA in tumors and metastatic lymph nodes and correlated it with counts of microvessel density. The expression of ECM1 (p = 0.004) and HA (p = 0.036) was significantly different between benign and malignant tumors. The expression of ECM1 was strongly associated with microvessel density (Spearman's rho = 0.513, p = 0.017), with the strongest expression present within caner nests. With respect to HA, expression was significantly associated with more advanced clinical TNM stage (Spearman's rho = 0.521, p = 0.015). The link was stronger in cases where metastasis or recurrence occurred than in those without complication (p = 0.014). No significant relationship was found betw...
Advances in experimental medicine and biology, 2007
Triptolide (TPL), a small molecule purified from the herb Tripterygium wilfordii, has potential c... more Triptolide (TPL), a small molecule purified from the herb Tripterygium wilfordii, has potential clinical application for suppression of chronic autoimmune disorders and inhibition of tumor growth. However, its mechanism of action is largely unknown. In this study, the effect of TPL on mitochondria was explored with a panel of molecular probes that detect the alteration of mitochondrial functions. When Lewis lung carcinoma (LLC) cells were treated with different doses of TPL for four hours, impaired mitochondrial functions were detected. This included an increased production of reactive oxygen species, the opening of the transition pore of mitochondria, the depolarization of the mitochondria membrane, the inhibition of the production of ATP and increased release of ATP as well as the induction of apoptosis. It is likely that by impairment of mitochondrial function, TPL exerts its inhibitory effect on growth of tumor and progression of inflammatory disease.
In this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine... more In this study, a hyaluronan-binding complex, which we termed Metastatin, was isolated from bovine cartilage by affinity chromatography and found to have both antitumorigenic and antiangiogenic properties. Metastatin was able to block the formation of tumor nodules in the lungs of mice inoculated with B16BL6 melanoma or Lewis lung carcinoma cells. Single i.v. administration of Metastatin into chicken embryos inhibited the growth of both B16BL6 mouse melanoma and TSU human prostate cancer cells growing on the chorioallantoic membrane. The in vivo biological effect may be attributed to the antiangiogenic activity because Metastatin is able to inhibit the migration and proliferation of cultured endothelial cells as well as vascular endothelial growth factor-induced angiogenesis on the chorioallantoic membrane. In each case, the effect could be blocked by either heat denaturing the Metastatin or premixing it with hyaluronan, suggesting that its activity critically depends on its ability ...
Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfecte... more Mice bearing primary tumors produced by LacZ-tagged MCF-7 human breast carcinoma cells transfected with fibroblast growth factor (FGF) 1 have frequent micrometastases, but macrometastases are not observed. i.v. injection of FGF-1-transfected tumor cells produced no pulmonary macrometastases, and removal of primary tumors resulted in the disappearance of spontaneous micrometastases. Thus, failure of micrometastases to proliferate was not due to inhibitory factors released from the primary tumor, and the presence of the primary tumor is required for maintenance of the micrometastases. This indicates that the micrometastases result from continued seeding from the primary tumor balanced by clearance from the metastatic site. Tumor emboli trapped in the vessels of lungs and lymph nodes and single tumor cells observed in the pulmonary vein implied that FGF-1-overexpressing MCF-7 cells are deficient in their ability to extravasate. The frequency of tumor cells incorporating bromodeoxyuridi...
A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediat... more A number of hyaluronan (HA) binding proteins such as soluble CD44, receptor for hyaluronan-mediated motility (RHAMM), and metastatin inhibit tumor growth and metastasis. To determine whether the HA binding motif is the element responsible for the antitumor effect of this family of proteins, we examined the biological activity of a 42-amino acid peptide (designated as BH-P) that contains three HA binding motifs [B(X(7))B] from human brain HA binding protein. In initial experiments with cultured cells, we found that synthetic BH-P inhibited the proliferation and colony formation of tumor cells. It also blocked the growth of tumors on the chorioallantoic membranes of 10-day chicken embryos. In addition, MDA-435 melanoma cells that had been transfected with an expression vector for BH-P grew at a slower rate in nude mice than the vector-alone transfectants. Final studies revealed that the BH-P could activate caspase-8, caspase-3, and poly(ADP-ribose) polymerase and trigger the apoptosis...
Increased extracellular matrix 1 (ECM1) expression, as determined by real-time reverse transcript... more Increased extracellular matrix 1 (ECM1) expression, as determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), has been recently proposed as a novel and independent diagnostic marker for malignant thyroid nodules. However ECM1 protein expression has not been previously evaluated in thyroid tumors. Real-time RT-PCR is not widely available and this has important implications for the widespread use of the expression of this gene for diagnosis. The purpose of the current study is to evaluate and compare ECM1 expression by real-time RT-PCR with immunohistochemistry (IHC) as diagnostic aids in thyroid neoplasms. ECM1 expression was studied in normal thyroid tissues (n = 14) and primary thyroid tumors (19 benign, 30 malignant) using both techniques. The performance characteristics of ECM1 expression were examined with receiver operating characteristic curves and the resulting area under the curve. ECM1 was highly expressed in thyroid carcinomas, compared with benign thyroid tissues, both at the mRNA (P < 0.01) and protein (P = 0.06) levels. However, ECM1 mRNA expression appeared to be a more sensitive marker of thyroid malignancy than IHC (AUC = 0.77 and 0.65, respectively). ECM1 expression by both methods was useful in identifying malignant follicular, but not Hürthle cell neoplasms. We confirm that ECM1 expression has potential utility as an independent diagnostic marker for thyroid cancer, although, performance characteristics were lower than previously published. Furthermore, IHC was not as sensitive as real-time RT-PCR in identifying malignant lesions. Prospective studies are needed to further clarify the role of ECM1 expression as a diagnostic marker.
Advances in Experimental Medicine and Biology, 2003
Breast tumors expressing no detectable FGFs (MCF-7) were compared with tumors transfected with FG... more Breast tumors expressing no detectable FGFs (MCF-7) were compared with tumors transfected with FGF4 or FGF1 (FGF4/MCF-7 or FGF1/MCF-7), and with MDA-MB-435, which produce endogenous FGF2. Tumor blood flow was measured by 133Xe diffusion, oxygen distribution was measured by Eppendorf pO2 histography, and vascular density was measured by CD31 staining. Tumors that overexpress angiogenic factors grew at a rate far exceeding that of MCF-7. The FGF producing tumors also had much higher metastatic rates to lung. Tumor blood flow was significantly higher in the two FGF-transfected xenografts compared with the parent MCF7. Median tumor pO2 was also higher, and tumor oxygenation was preserved even for large tumors. The vascular density as determined by CD31 staining, however, was not markedly increased in tumors overexpressing angiogenic factors. We found that angiogenic factors preserve and augment neovascular function, thus facilitating tumor growth and progression.
Advances in experimental medicine and biology, 2013
Tumor hypoxia is probably the most important not yet measurable factor that predicts the outcome ... more Tumor hypoxia is probably the most important not yet measurable factor that predicts the outcome of cancer therapy. Hypoxic tumors are resistant to radiation, chemotherapy, and surgery. They signal tumor cells to grow, invade, survive cytotoxic-factor assault, and increase metastatic activity. Therapies aimed at reversing hypoxia-related treatment resistance or normalizing hypoxia are proven effective with level 1 evidence. The weak link remains the lack of satisfactory methods of measurement of tumor oxygenation.
Advances in Experimental Medicine and Biology, 2005
Hyaluronan (HA), a large negatively-charged polysaccharide, is a major component of vessel basal ... more Hyaluronan (HA), a large negatively-charged polysaccharide, is a major component of vessel basal membrane. HA is expressed by a variety of cells, including tumor and endothelial cells. We hypothesized that HA could be up-regulated by hypoxia to enhance vessel formation. To determine the effect of hypoxia on the production of HA, tumor cells were treated with either media alone (control) or a hypoxia inducer (CoCl or NaN3) for 24 h. The level of HA in the media was then measured by ELISA. The results showed that both CoCl and NaN3 induced the production of HA. Since the low molecular weight form of HA (SMW) possesses pro-angiogenic properties, we investigated whether hypoxia-induced HA can be processed into SMW. Under hypoxic conditions, the activity of hyaluronidase, the enzyme responsible for degrading HA, was measured by an ELISA-like assay. The activity of hyaluronidase was shown to be up-regulated by hypoxia and, further, could carry out the function of processing HA into SMW. In addition, the hypoxic areas of tumor tissues were stained strongly with biotinylated HA-binding proteins, indicating that the level of HA was high compared to the oxic areas. This study demonstrates that hypoxia can stimulate the production of HA and the activity of hyaluronidase, which may promote angiogenesis as a compensation mechanism for hypoxia.
Chick chorioallantoic membrane (CAM) angiogenesis assay has been widely used for finding drugs ta... more Chick chorioallantoic membrane (CAM) angiogenesis assay has been widely used for finding drugs targeting new blood vessel development in cancer research. In addition to the setup materials and protocols, laboratory findings depend on the quantification and analysis of microscopic blood vessel images. However, it is still a challenging problem because of the high complexity of blood vessel branching structures. We applied preprocessing on CAM microscopic images by keeping the integrity of minor branches in the vessel structure. We then proposed an efficient way to automatically extract blood vessel centerlines based on vector tracing starting from detected seed points. Finally, all branches were coded to construct an abstract model of the branching structure, which enabled more accurate modeling for in-depth analysis. The framework was applied in quantifying Icaritin (ICT) inhibition effects on angiogenesis in a CAM model. Experimental results showed the high accuracy in blood vessel...
Mitochondria play pivotal roles in cellular handling of oxygen and in apoptosis, the ordered suic... more Mitochondria play pivotal roles in cellular handling of oxygen and in apoptosis, the ordered suicide response of cells to irradiation. The involvement of expression products from the 16.5 kb human mitochondrial genome in these activities has been studied widely. However, little is known about effects of irradiation on mammalian mitochondrial DNA (mtDNA). The relative lack of mtDNA repair mechanisms compared with nuclear DNA (nDNA) predicts particular vulnerability to irradiation. Using a technique developed to ascertain mtDNA:nDNA ratios, we previously showed that this ratio increases dramatically in murine small bowel within 48 hours following whole body irradiation. We now report that those levels continue to rise for four days and remain elevated at close to that level beyond 30 days after 5 Gy of irradiation.We further demonstrate that levels of the mtDNA-specific DNA polymerase-γ (Pol-γ ) also show a sharp and sustained increase during this time course after a 2-Gy dose. Paradoxically, transcription factor A (TFAM), exhibited the directly opposite response.
Advances in experimental medicine and biology, 2009
The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondr... more The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclu...
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Papers by Lurong Zhang