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Growth inhibition of esophageal cancer cells by extracellular nucleotides
A534 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
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COMPARISON OF KI-RAS MUTATION, CELL PROLIFERATION, AND APOPTOSIS BETWEEN 2 DISTINCT MORPHOLOGIC TYPES OF COLORECTAL TUMOR. Masahiro Ikegami, Hiroyuki Takahashi, Tetsuya Etou, Shigeo Koido, Hiroshi Asakawa, Mika Anami, Yoshiya Miyagawa, Akira Torii, Gotaro Toda, Jikei Univ Sch of Medicine, Tokyo, Japan. Colorectal adenoma and early carcinoma are morphologically classified as 2 distinct subtypes: protruded type (PT) and flat type (FT). PT-tumor has been known to develop associated with adenoma-carcinoma sequence including Ki-ras mutation. Progress of colorectal endoscopy technique for last several years has made it feasible to find small flat lesions in the mucosa and such lesions has been admitted as FT tumor. FT tumor may have a potential as an early lesion of invasive carcinoma that has been thought to develop "de novo". Under the hypothesis that different genetic and/or epi-genetic events occur during tumor progressions between PT and FT, we compared frequency of Ki-ras mutation, rate of proliferating cells and apoptotic cells between these 2 types of colorectal tumor. Seventy colorectal adenoma and carcinoma (33 PT, 37 FT) were obtained by endoscopic polypectomy or mucosal resection. They were histologically subclassified as adenoma with low-grade atypia (ALA), adenoma with high grade atypia(AHA), intramucosal carcinoma(IMC), and sub-mucosal invasive carcinoma (SMC). PCR-based dot blot hybridization was performed to detect Ki-ras codon 12 mutations. To detect cell proliferation and apoptosis, immunostaining with Ki-67 and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) method were performed. The percentage of positive cells were described as Ki-67 labeling index (Ki-LI) and TUNEL labeling index (TUNEL-LI) in each layer (upper 1/3, middle 113, and lower 113) and total. In PT, Ki-ras mutation at codon 12 was detected in 25% of AHA, 37.5% in IMC, and 31.3% in SMC. In FT, it was detected in 0%, 25%, 0%, respectively. In adenoma (ALA and AHA), Ki-LI of upper layer in PT were significantly higher than those in FT(p=0.OO7). Ki-LI of lower layer in FT-SMC were significantly higher than those in PT-SMC(P=0.04). TUNEL-LI of FT-SMC, though not statistically significant, were lower than those of PT-SMC in all of the layers. FT tumors may not need Ki-ras mutation for progression unlike PT tumors do. Higher cell proliferation in the upper layer of PT-adenoma associates with the morphological protuberance of the tumor. Higher cell proliferation rate and tendency of less apoptotic cellsin the lower layer of FT-SMC suggest more aggressive nature in the specific tumor type. Thus, different genetic and epi-genetic events may associate with differences of their morphological features and biological characters between those 2 types of colorectal tumor.
INVOLVEMENT OF CASPASE-3 ACTIVATION AND P53 IN ARSENIC TRIOXIDE INDUCED APOPTOSIS IN HUMAN GASTRIC CANCER CELLS. Xh Jiang, Sk Lam, Sh Jiang, St Yuen, Ch Cho, Kc Lai, Bey Wong, Dept of Medicine, Univ of Hong Kong, Hong Kong; Dept of Gastroenterology, Rui-jin Hosp, Shanghai, P. R. China; Dept of Pathology, Univ of Hong Kong, Hong Kong; Dept of Pharmacology, Univ of Hong Kong, Hong Kong. Background: Arsenic trioxide (ASZ0 3) can induce clinical remission in patients suffering from acute promyelocytic leukemia. In vitro studies showed it could induce apoptosis in leukemia cell lines through the activation of caspases. We investigated the potential use of (ASZ0 3)on human gastric cancer and its possible mechanisms. Method: Human gastric cancer cell AGS was treated with various concentrations (0.1 to 100 p,M) of (ASZ0 3 ) for 24-72 hrs. Cell growth was assessed by MTT assay and apoptosis was determined by acridine orange staining, flow cytometry and DNA gel electrophoresis. Protein levels of p53, p21, c-rnyc, bcl-2 and bax were detected by western blot. Caspase-3 activity was detected by using its colorimetric substrate DEVD-pNA and cleavage of PARP. Results: AS Z0 3 inhibited cell growth in AGS cells following a dose-dependent manner. Further, AS Z0 3 iniduced apoptosis in AGS cells after 24 hr treatment with concentrations ranging from 0.5 to 10 p.M. However, there was no alteration of the cell cycle phase distribution during the whole period. In addition, western blot analysis revealed that treatment with I p.M AS Z 0 3 resulted in marked increase in p53 protein level as early as 2 hr and reached a maximum at 12 h, which was approximately 3 times of control. However, there was no changes of protein level in other apoptosis-related genes, i.e. p2l, c-myc, bcl-2 and bax, during treatment. Coincubation with p53 antisense oligonucleotide significantly suppressed ASzO}"induced intracellular p53 overexpression and apoptosis (5.9% in co-treatment vs. 19.5% in AS Z03 treatment alone). AS Z03 treatment increased the activity of caspase-3 which was in parallel with the percentage increase in apoptosis, and western blot analysis showed the cleavage of caspase-3 and its substrate PARP. The caspase inhibitors (zVAD-fmk and zDEVD-fmk)suppressed AS Z0 3 -induced caspase-3 activation and apoptosis. Conclusions: AS Z03 inhihited cell growth and induced apoptosis in gastric cancer cells. p53 played a crucial role. The apoptotic pathway involved activation of caspase-3 protease and PARP degradation.
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GROWTH INHIBITION OF ESOPHAGEAL CANCER CELLS BY EXTRACELLULAR NUCLEOTIDES. Havva Kap, Michael Hoepfner, Kerstin Maaser, Ernst-Otto Riecken, Hans Scheruebl, Benjamin Franklin Clinics, Free Univ of Berlin, Berlin, Germany. Introduction: Extracellular ATP is known to inhibit cancer growth in various tumor models by activating specific purinergic receptors (P2receptors), which alter intracellular [Caz+]j' Since the incidence of esophageal cancer rises and since its therapy remains unsatisfying, new therapeutic approaches are mandatory. Thus, we investigated the functional expression and possible antiproliferative effects of P2-purinergic receptors in esophageal cancer cells. Methods: The human esophageal squamous cancer cell line KYSE-140 and the human esophageal adenocarcinoma cell line OE-33 were used as models. Functional expression of purinergic receptors was evaluated by means of [Caz+];-imaging (FURA2-method). Cell proliferation was determined by the crystal violet-assay. Results: In both cell lines, application of various extracellular nucleotides led to a dose-dependent, biphasic rise in [Caz+L. The rank order of potency was ATP= UTP>ATPoyS>ADP= UDP> >2-methylthio-ATP. a,/3-methyleneATP, a specific agonist for ionotropic P2-receptors was ineffective. Moreover, complete cross-desensitization between the equipotent agonists ATP and UTP was observed, indicating that both ligands acted competitively at the same binding-site. These pharmacological features strongly suggest the expression of G-protein coupled P2Yz-purinergic receptors. The biphasic rise of [Caz+L consisted of an initial peak caused by intracellular Caz+ mobilisation from the ER and a subsequent plateau phase due to transmembranous Caz+-influx. The Caz+-influx was proven by experiments with the Ca z+-ATPase inhibitor thapsigargin and by withdrawal of extracellular Ca2+. The [Ca2+]j elevation was partially inhibited by pertussis toxin (PTX) pretreatment. Moreover, the PLC inhibitor U73122 dosedependently reduced the [Caz+L signal, indicating the involvement of both PTX-insensitive Gq - and of PTX-sensitive Gi-proteins each linked to PLC activation. Prolonged activation of P2Y2 receptors by ATP or its stable analog ATPoyS dose-dependently inhibited cell proliferation. When ATPoyS was combined with the cytostatic drug 5-FU, synergistic antiproliferative effects were observed. Conclusions: Our study shows that esophageal cancer cells express functional P2Y Z receptors whose sustained activation results in growth inhibition and is capable of potentiating anti-neoplastic effects of 5-FU. Thus, P2Yz receptors are promising target proteins for new therapeutic approaches in the treatment of esophageal cancer.
APOPTOSIS INDUCED BY TRIPTOLIDE IN HUMAN GASTRIC CANCER IS DEPENDENT ON THE CONSTITUTIVE EXPRESSION OF WILD TYPE P53 AND ACTIVATION OF CASPASES. Xh Jiang, Sk Lam, D. Yang, Sh Jiang, St Yuen, Ch Cho, Kc Lai, Bey Wong, Dept of Medicine, Univ of Hong Kong, Hong Kong; Dept of Chemistry, Univ of Hong Kong, Hong Kong; Dept of Gastroenterology, Rui-jin Hosp, Shanghai, P. R. China; Dept of Pathology, Univ of Hong Kong, Hong Kong; Dept of Pharmacology, Univ of Hong Kong, Hong Kong. Background: Extracts of the Chinese herbal remedy Tripterygium wilfordii Hook f (TWHf) have been reported to be effective in the treatment of patients with a variety of inflammatory and autoimmune diseases for centuries. Triptolide is the major active component in the extract, recently it was also found to have significant antineoplastic effect. In the present study, we investigated the effect and possible mechanisms of triptolide in human gastric cancer cell lines. Method: Two gastric cancer cell lines, MKN-28 (mutant p53) and AGS (wild-type p53), were compared as to growth inhibition, apoptosis, cell cycle, apoptosis-related gene expression and caspase activity in response to triptolide treatment. Cell growth was measured by MTT assay. Apoptosis was characterized by acridine orange staining, DNA fragmentation, and cell cycle kinetics by flow cytometry. The protein level of p53, p21 wafl/co p ] , c-myc, bcl-2 and bax were determined by Western blotting. Caspase-3 activity was detected by using its colorimetric substrate DEVD-pNA and cleavage of PARP. Results: Triptolide initiated growth inhibition and apoptosis in both cell lines, although AGS was more sensitive to the growth inhibitory activity and induction of apoptosis. AGS cells were arrested in the GoIG] phase after 24h treatment, whereas MKN-28 showed no cell cycle alteration during the whole period of treatment. Triptolide induced the protein expression of the p53 gene and its target genes, p21 wafIrcip l and bax only in the AGS cells. The role of p53 was further confirmed by the result that down-regulation of p53 with its antisense oligonucleotide suppressed triptolide induced apoptosis in AGS cells. In addition, triptolide increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the AGS cells which was in parallel with the increase of apoptosis. The caspase inhibitors (zVAD-fmk and zDEVD-fmk) suppressed triptolide induced caspase activation and apoptosis in both cell lines. Conclusions: These results suggest that triptolide-induced apoptosis is more extensive in gastric cancer cells that express wild-type R53, possibly due to the involvement of the p53 regulated genes p21 wa l ycip l and bax although triptolide can also induce apoptosis by means of a p53-independent mechanism. Both pathways of triptolide-induced apoptosis appear to involve activation of caspases leading to a caspase-dependent cell death process.
2828
GASTROENTEROLOGY Vol. 118, No.4
2825
2827
COMPARISON OF KI-RAS MUTATION, CELL PROLIFERATION, AND APOPTOSIS BETWEEN 2 DISTINCT MORPHOLOGIC TYPES OF COLORECTAL TUMOR. Masahiro Ikegami, Hiroyuki Takahashi, Tetsuya Etou, Shigeo Koido, Hiroshi Asakawa, Mika Anami, Yoshiya Miyagawa, Akira Torii, Gotaro Toda, Jikei Univ Sch of Medicine, Tokyo, Japan. Colorectal adenoma and early carcinoma are morphologically classified as 2 distinct subtypes: protruded type (PT) and flat type (FT). PT-tumor has been known to develop associated with adenoma-carcinoma sequence including Ki-ras mutation. Progress of colorectal endoscopy technique for last several years has made it feasible to find small flat lesions in the mucosa and such lesions has been admitted as FT tumor. FT tumor may have a potential as an early lesion of invasive carcinoma that has been thought to develop "de novo". Under the hypothesis that different genetic and/or epi-genetic events occur during tumor progressions between PT and FT, we compared frequency of Ki-ras mutation, rate of proliferating cells and apoptotic cells between these 2 types of colorectal tumor. Seventy colorectal adenoma and carcinoma (33 PT, 37 FT) were obtained by endoscopic polypectomy or mucosal resection. They were histologically subclassified as adenoma with low-grade atypia (ALA), adenoma with high grade atypia(AHA), intramucosal carcinoma(IMC), and sub-mucosal invasive carcinoma (SMC). PCR-based dot blot hybridization was performed to detect Ki-ras codon 12 mutations. To detect cell proliferation and apoptosis, immunostaining with Ki-67 and terminal deoxynucleotidyl transferasemediated dUTP nick end labeling (TUNEL) method were performed. The percentage of positive cells were described as Ki-67 labeling index (Ki-LI) and TUNEL labeling index (TUNEL-LI) in each layer (upper 1/3, middle 113, and lower 113) and total. In PT, Ki-ras mutation at codon 12 was detected in 25% of AHA, 37.5% in IMC, and 31.3% in SMC. In FT, it was detected in 0%, 25%, 0%, respectively. In adenoma (ALA and AHA), Ki-LI of upper layer in PT were significantly higher than those in FT(p=0.OO7). Ki-LI of lower layer in FT-SMC were significantly higher than those in PT-SMC(P=0.04). TUNEL-LI of FT-SMC, though not statistically significant, were lower than those of PT-SMC in all of the layers. FT tumors may not need Ki-ras mutation for progression unlike PT tumors do. Higher cell proliferation in the upper layer of PT-adenoma associates with the morphological protuberance of the tumor. Higher cell proliferation rate and tendency of less apoptotic cellsin the lower layer of FT-SMC suggest more aggressive nature in the specific tumor type. Thus, different genetic and epi-genetic events may associate with differences of their morphological features and biological characters between those 2 types of colorectal tumor.
INVOLVEMENT OF CASPASE-3 ACTIVATION AND P53 IN ARSENIC TRIOXIDE INDUCED APOPTOSIS IN HUMAN GASTRIC CANCER CELLS. Xh Jiang, Sk Lam, Sh Jiang, St Yuen, Ch Cho, Kc Lai, Bey Wong, Dept of Medicine, Univ of Hong Kong, Hong Kong; Dept of Gastroenterology, Rui-jin Hosp, Shanghai, P. R. China; Dept of Pathology, Univ of Hong Kong, Hong Kong; Dept of Pharmacology, Univ of Hong Kong, Hong Kong. Background: Arsenic trioxide (ASZ0 3) can induce clinical remission in patients suffering from acute promyelocytic leukemia. In vitro studies showed it could induce apoptosis in leukemia cell lines through the activation of caspases. We investigated the potential use of (ASZ0 3)on human gastric cancer and its possible mechanisms. Method: Human gastric cancer cell AGS was treated with various concentrations (0.1 to 100 p,M) of (ASZ0 3 ) for 24-72 hrs. Cell growth was assessed by MTT assay and apoptosis was determined by acridine orange staining, flow cytometry and DNA gel electrophoresis. Protein levels of p53, p21, c-rnyc, bcl-2 and bax were detected by western blot. Caspase-3 activity was detected by using its colorimetric substrate DEVD-pNA and cleavage of PARP. Results: AS Z0 3 inhibited cell growth in AGS cells following a dose-dependent manner. Further, AS Z0 3 iniduced apoptosis in AGS cells after 24 hr treatment with concentrations ranging from 0.5 to 10 p.M. However, there was no alteration of the cell cycle phase distribution during the whole period. In addition, western blot analysis revealed that treatment with I p.M AS Z 0 3 resulted in marked increase in p53 protein level as early as 2 hr and reached a maximum at 12 h, which was approximately 3 times of control. However, there was no changes of protein level in other apoptosis-related genes, i.e. p2l, c-myc, bcl-2 and bax, during treatment. Coincubation with p53 antisense oligonucleotide significantly suppressed ASzO}"induced intracellular p53 overexpression and apoptosis (5.9% in co-treatment vs. 19.5% in AS Z03 treatment alone). AS Z03 treatment increased the activity of caspase-3 which was in parallel with the percentage increase in apoptosis, and western blot analysis showed the cleavage of caspase-3 and its substrate PARP. The caspase inhibitors (zVAD-fmk and zDEVD-fmk)suppressed AS Z0 3 -induced caspase-3 activation and apoptosis. Conclusions: AS Z03 inhihited cell growth and induced apoptosis in gastric cancer cells. p53 played a crucial role. The apoptotic pathway involved activation of caspase-3 protease and PARP degradation.
2826
GROWTH INHIBITION OF ESOPHAGEAL CANCER CELLS BY EXTRACELLULAR NUCLEOTIDES. Havva Kap, Michael Hoepfner, Kerstin Maaser, Ernst-Otto Riecken, Hans Scheruebl, Benjamin Franklin Clinics, Free Univ of Berlin, Berlin, Germany. Introduction: Extracellular ATP is known to inhibit cancer growth in various tumor models by activating specific purinergic receptors (P2receptors), which alter intracellular [Caz+]j' Since the incidence of esophageal cancer rises and since its therapy remains unsatisfying, new therapeutic approaches are mandatory. Thus, we investigated the functional expression and possible antiproliferative effects of P2-purinergic receptors in esophageal cancer cells. Methods: The human esophageal squamous cancer cell line KYSE-140 and the human esophageal adenocarcinoma cell line OE-33 were used as models. Functional expression of purinergic receptors was evaluated by means of [Caz+];-imaging (FURA2-method). Cell proliferation was determined by the crystal violet-assay. Results: In both cell lines, application of various extracellular nucleotides led to a dose-dependent, biphasic rise in [Caz+L. The rank order of potency was ATP= UTP>ATPoyS>ADP= UDP> >2-methylthio-ATP. a,/3-methyleneATP, a specific agonist for ionotropic P2-receptors was ineffective. Moreover, complete cross-desensitization between the equipotent agonists ATP and UTP was observed, indicating that both ligands acted competitively at the same binding-site. These pharmacological features strongly suggest the expression of G-protein coupled P2Yz-purinergic receptors. The biphasic rise of [Caz+L consisted of an initial peak caused by intracellular Caz+ mobilisation from the ER and a subsequent plateau phase due to transmembranous Caz+-influx. The Caz+-influx was proven by experiments with the Ca z+-ATPase inhibitor thapsigargin and by withdrawal of extracellular Ca2+. The [Ca2+]j elevation was partially inhibited by pertussis toxin (PTX) pretreatment. Moreover, the PLC inhibitor U73122 dosedependently reduced the [Caz+L signal, indicating the involvement of both PTX-insensitive Gq - and of PTX-sensitive Gi-proteins each linked to PLC activation. Prolonged activation of P2Y2 receptors by ATP or its stable analog ATPoyS dose-dependently inhibited cell proliferation. When ATPoyS was combined with the cytostatic drug 5-FU, synergistic antiproliferative effects were observed. Conclusions: Our study shows that esophageal cancer cells express functional P2Y Z receptors whose sustained activation results in growth inhibition and is capable of potentiating anti-neoplastic effects of 5-FU. Thus, P2Yz receptors are promising target proteins for new therapeutic approaches in the treatment of esophageal cancer.
APOPTOSIS INDUCED BY TRIPTOLIDE IN HUMAN GASTRIC CANCER IS DEPENDENT ON THE CONSTITUTIVE EXPRESSION OF WILD TYPE P53 AND ACTIVATION OF CASPASES. Xh Jiang, Sk Lam, D. Yang, Sh Jiang, St Yuen, Ch Cho, Kc Lai, Bey Wong, Dept of Medicine, Univ of Hong Kong, Hong Kong; Dept of Chemistry, Univ of Hong Kong, Hong Kong; Dept of Gastroenterology, Rui-jin Hosp, Shanghai, P. R. China; Dept of Pathology, Univ of Hong Kong, Hong Kong; Dept of Pharmacology, Univ of Hong Kong, Hong Kong. Background: Extracts of the Chinese herbal remedy Tripterygium wilfordii Hook f (TWHf) have been reported to be effective in the treatment of patients with a variety of inflammatory and autoimmune diseases for centuries. Triptolide is the major active component in the extract, recently it was also found to have significant antineoplastic effect. In the present study, we investigated the effect and possible mechanisms of triptolide in human gastric cancer cell lines. Method: Two gastric cancer cell lines, MKN-28 (mutant p53) and AGS (wild-type p53), were compared as to growth inhibition, apoptosis, cell cycle, apoptosis-related gene expression and caspase activity in response to triptolide treatment. Cell growth was measured by MTT assay. Apoptosis was characterized by acridine orange staining, DNA fragmentation, and cell cycle kinetics by flow cytometry. The protein level of p53, p21 wafl/co p ] , c-myc, bcl-2 and bax were determined by Western blotting. Caspase-3 activity was detected by using its colorimetric substrate DEVD-pNA and cleavage of PARP. Results: Triptolide initiated growth inhibition and apoptosis in both cell lines, although AGS was more sensitive to the growth inhibitory activity and induction of apoptosis. AGS cells were arrested in the GoIG] phase after 24h treatment, whereas MKN-28 showed no cell cycle alteration during the whole period of treatment. Triptolide induced the protein expression of the p53 gene and its target genes, p21 wafIrcip l and bax only in the AGS cells. The role of p53 was further confirmed by the result that down-regulation of p53 with its antisense oligonucleotide suppressed triptolide induced apoptosis in AGS cells. In addition, triptolide increased the activity of caspase in both cell lines, however, the effect was much more pronounced in the AGS cells which was in parallel with the increase of apoptosis. The caspase inhibitors (zVAD-fmk and zDEVD-fmk) suppressed triptolide induced caspase activation and apoptosis in both cell lines. Conclusions: These results suggest that triptolide-induced apoptosis is more extensive in gastric cancer cells that express wild-type R53, possibly due to the involvement of the p53 regulated genes p21 wa l ycip l and bax although triptolide can also induce apoptosis by means of a p53-independent mechanism. Both pathways of triptolide-induced apoptosis appear to involve activation of caspases leading to a caspase-dependent cell death process.
2828