- Email: [email protected]
Enhanced bax gene expression reduces cell growth and sensitizes human gastric cancer cells to chemotherapeutic agents
AGAA535
April 2000
2829
2831
IGFAXIS, PROLIFERATION, APOPTOSIS AND COLORECTAL ADENOMA RISK. Temitope O. Keku, Joseph A. Galanko, Neha P. Mehta, Anita P. Terse, John T. Woosley, Pauline K. Lund, Robert S. Sandler, Univ of North Carolina, Chapel Hill, NC.
ENHANCED BAX GENE EXPRESSION REDUCES CELL GROWTH AND SENSITIZES HUMAN GASTRIC CANCER CELLS TO CHEMOTHERAPEUTIC AGENTS. Koga Komatsu, Jun-ichi Miyazaki, Susumu Suzuki, Michiro Otaka, Sumio Watanabe, Tooru Shimosegawa, Takayoshi Toyota, Akita Univ Sch of Medicine, Akita, Japan; Osaka Univ Med Sch, Suita, Japan; Tohoku Univ Sch of Medicine, Sendai, Japan. An increased understanding of the genes involved in the regulation of apoptosis has led to the hypothesis that dysregulation of apoptosis underlies tumor development, progression, and its resistance to therapies. Therapies directed at altering the levels of expression of apoptosis-regulating genes may be a useful strategy for cancer therapy. Therefore, it is important to know the effects of modulating these genes in cancer cells. Bax, an inducer of apoptosis, counters the death repressor activity of Bcl-2. Overexpression of Bax has led to increased cell death in various cancer cells. The bax gene is known as a primary response gene of p53, and has been shown to have a tumor suppressor function as well as to carry somatic mutations in certain cancers, including gastric cancer. In this study, we investigated the effect of Bax overexpression on cell proliferation and sensitivity to chemotherapeutic agents in two gastric cancer cells using a CrelloxP-mediated expression system. Methods: We transfected bax-ainto MKN-28 and MKN-45 under the control of an inducible CrelloxP expression system, and stably transfected cells were obtained. Bax expression was induced by infection of a Cre expressing adenoviral vector. Cell viability after induction of bax was measured by the MTT assay. We also examined the cell viability of MKN-28, pre- and post-induction of bax, under some chemotherapeutic agents, and calculated the concentration of each drug yielding 50% inhibition of cell growth. Results: After induction of bax, both cells showed decreased growth rate within 2 days (in MKN28) and 3 days (in MKN-45), compared with infecting LacZ-expressing adenoviral vector or a mock infection, partially due to increased apoptosis. This death-promoting effect of bax was also shown by transient cotransfection assay, a bax-expression plasmid or empty vector with a lacZ expression plasmid into parental MKN-45 cells. The number of lacZpositive cells were markedly reduced in bax-transfected cells (0.2:':0.3% vs. 12.2:':2.1 %,p
Insulin like growth factors (IGFs) are mitogenic for normal and neoplastic cells. Plasma IGF-I levels correlate with the risk of prostate, lung and breast cancer. Acromegalies have high levels ofIGF-I and growth hormone (GH), and show at increased risk of colorectal cancer. We examined the relationship between plasma IGF-I, IGF-ll, IGFBP-3, crypt cell proliferation and apoptosis in an ongoing study of colorectal adenoma risk factors. Methods: Blood and biopsy samples of normal colon were obtained from consenting adult subjects referred to the University of North Carolina Hospitals for colonoscopy. Plasma IGFs and IGFBP-3 were measured by ELISA. Proliferation and apoptosis were measured by whole crypt mitotic counts morphologyrrUNEL respectively. Results: 582 subjects (203 adenomas cases; 35% and 379 none adenoma controls; 65%) were assessed. Comparing cases to controls, there were no statistically significant differences in mean plasma IGF-I, IGF-II and IGFBP-3 levels, and no significant differences in proliferation. Cases had lower apoptosis compared to the controls (p< 0.(01). Consistent with prior studies cases also had significantly higher waist/hip ratio, were more likely to be men and were older. We therefore used logistic regression to examine the association between adenoma status and IGF-I, IGF-II and IGFBP-3 while controlling for age, gender, body mass index (BMI) and smoking. In the multivariate models, only age and gender were significant predictors of adenoma status. Compared to the lowest quartile of IGF-I, those in the highest quartile were not more likely to have an adenoma (adjusted odds ratio [OR] 0.80, 95% CI 0.36-1.76). Similarly, those in the highest quartile of IGF-II were not more likely to have adenomas (OR 0.66, 95% CI 0.26-1.70). Among the cases, insulin, IGF-I, female sex and higher waist/hip ratio were associated with increased proliferation. Only BMI was associated with increased proliferation among the controls. Among all subjects being a case, having elevated IGF-I, IGF-II levels, high waist/hip ratio and female sex were associated with low apoptosis. Elevated levels of IGFBP-3 were associated with increased apoptosis among all the subjects. Conclusions: Plasma IGF-I, IGF-II and IGFBP-3, and proliferation did not correlate with the presence of adenomas. Low levels of apoptosis in the normal colonic mucosa were associated with adenomas indicating that this could be a predictor of adenoma risk.
2830
2832
PROLONGED ACTIVATION OF MITOGEN·ACTIVATED PRO· TEIN KINASES (MAPKS) IN NSAIDS·INDUCED APOPTOSIS. Won Ho Kim, Soo Hyun Jin, Eun Hyae Kang, Tae II Kim, Jin Kyung Kang, Yonsei Univ Coli of Medicine, Seoul, South Korea.
INHIBITION OF APOPTOSIS BY GROWTH FACTORS AND PROSTAGLANDIN· A KEY EVENT IN HEALING OF STRESS DAMAGE? Peter Ch Konturek, Pierzchalski Piotr, Tomasz Brzozowski, Alexandra Szlachcic, Stanislaw J. Konturek, Eckhart G. Hahn, Dept Med I , Univ Med, Erlangen, Germany; Dept Physiol , Univ Med Sch, Cracow, Poland. Growth factors such as epidermal growth factor (EGF) and pancreatic trypsin inhibitor (PSTI) are involved in healing of gastric lesions induced by stress but their influence on apoptosis in the gastric mucosa exposed to stress remains unknown. In this study we compared the ulcer healing effects of EGF and PSTI (50 ILglkgi.p.) with those exhibited by 16,16 dm PGE 2 (5 ILglkgi.p.), and we determined the alterations in apoptosis, gastric blood flow (GBF) and expression of cytokines in rats exposed to 3.5 h of water immersion and restraint stress (WRS). The animals were killed immediately (time 0) and at 3, 6, 12,24 and 48 h after the end ofWRS and the area of gastric lesions was measured by planimetry and gastric blood flow (GBF) was determined by H2-gas clearance method. Blood was collected for measurement of plasma IL-l{3 and TNFa levels by ELISA and mucosal biopsy samples were taken for determination of IL-l {3 and TNFa mRNA and expression of apoptosis-releted proteins such as Bax and Bcl-2 mRNA by RT-PCR and Southern blot. The rate of apoptosis was measured by transferase-mediated dUPT-biotin nick end-labeling (TUNEL) method. After 3.5 h of exposure to WRS gastric erosions occurred and their number reached the maximum at 12 and then declined at 24 h to disappear at 48 h. The GBF was significantly decreased immediately after WRS but then it rose progressively starting from 3 up to 48 h after WRS. Expression of IL-l{3 and TNFa mRNA rose up to 12 h then declined at 24 h and returned to the control level at 48 h after WRS and this was accompanied by the rise in plasma IL-I{3 and TNFa levels. Bax and Bcl-2 mRNA were undetectable in the intact gastric mucosa but at 6 h after WRS, a significant rise in the number of apoptotic cells (TUNEL method) and an imbalance between Bax and Bcl-2 (BaxlBcl-2 ratioz l) were observed. Bax mRNA gradually increased in first 4 hours to decline at 24 h after WRS while Bcl-2 was detected at 6 and 12 h to decline to the control level at 24 h after WRS. Treatment with EGF, PSTI and PGE 2 accelerated significantly healing of WRS lesions, counteracted the imbalance between BaxIBcl-2 proteins and attenuated the rise in apoptosis and plasma IL-l {3 and TNFa levels after WRS. We conclude that: l)stress lesions involve overexpression of inflammatory cytokines, the fall in the GBF and enhancement in apoptosis triggered by the shift from cell death effector Bax to the cell death repressor Bcl-2 and 2)growth factors and PG may contribute to the recovery from WRS lesions by the attenuation of apoptosis and suppression of cytokine expression and release.
Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of and mortality from colon cancer. The mechanisms of the anti-neoplastic effect of NSAIDs are still far from complete understanding, but one of the possible mechanisms is an induction of apoptosis. We have previously reported the role of caspase-3, a key enzyme for the execution of apoptosis, in NSAIDs-induced apoptosis of colon cancer cells. Mitogenactivated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (1NK)/stress-activated protein kinase (SAPK) and p38 kinase are activated in response to different stimuli, have different downstream targets and, therefore, perform different functions. Of these MAPKs, JNKlSAPK and p38K are considered as important mediators of apoptotic signals in many systems. Aim: The aim of this study was to demonstrate the activation of MAPKs in NSAIDsinduced apoptosis of colon cancer cells and to elucidate a pathway leading to NSAIDs-induced apoptosis. Subjects and Methods: Apoptosis was detected and confirmed by confocal microscopic observation of chromatin condensation after DAPI stain and the demonstration of DNA fragmentation in agarose gel electrophoresis. Cell death was measuredd by trypan blue dye exclusion method. Caspase-3 activity was measured using tetrapeptide fluorogenic substrate, Ac-DEVD-AMC. MAPKs activation was assessed by Western blot using phospho-specific antibodies to MAPKs. Kinase assay using ATF-2 fusion protein as a substrate was also performed for the measurement of p38K activity. For the inhibition of p38K, pyridinylimidazole compound, SB203580, was utilized. Results: In the colon cancer cell line, HT-29, NSAIDs such as indomethacin, sulindac and nabumetone, induced dose- and time-dependent apoptosis as well as prolonged activation of MAPKs including ERK, JNK and p38K. SB203580, a p38K inhibitor, attenuated not only indomethacin-induced cell death but also caspase-3 activation. However, caspase inhibitors, such as Ac-DEVDCHO, Ac-YVAD-CHO and Z-VAD-FMK, did not affect p38K activation in indomethacin-treated HT-29 cells. Conclusion: From these results, we concluded that prolonged activation of MAPKs including p38K, followed by caspase-3 activation, is an important signal pathway mediating NSAIDs-induced colon cancer cell apoptosis.
April 2000
2829
2831
IGFAXIS, PROLIFERATION, APOPTOSIS AND COLORECTAL ADENOMA RISK. Temitope O. Keku, Joseph A. Galanko, Neha P. Mehta, Anita P. Terse, John T. Woosley, Pauline K. Lund, Robert S. Sandler, Univ of North Carolina, Chapel Hill, NC.
ENHANCED BAX GENE EXPRESSION REDUCES CELL GROWTH AND SENSITIZES HUMAN GASTRIC CANCER CELLS TO CHEMOTHERAPEUTIC AGENTS. Koga Komatsu, Jun-ichi Miyazaki, Susumu Suzuki, Michiro Otaka, Sumio Watanabe, Tooru Shimosegawa, Takayoshi Toyota, Akita Univ Sch of Medicine, Akita, Japan; Osaka Univ Med Sch, Suita, Japan; Tohoku Univ Sch of Medicine, Sendai, Japan. An increased understanding of the genes involved in the regulation of apoptosis has led to the hypothesis that dysregulation of apoptosis underlies tumor development, progression, and its resistance to therapies. Therapies directed at altering the levels of expression of apoptosis-regulating genes may be a useful strategy for cancer therapy. Therefore, it is important to know the effects of modulating these genes in cancer cells. Bax, an inducer of apoptosis, counters the death repressor activity of Bcl-2. Overexpression of Bax has led to increased cell death in various cancer cells. The bax gene is known as a primary response gene of p53, and has been shown to have a tumor suppressor function as well as to carry somatic mutations in certain cancers, including gastric cancer. In this study, we investigated the effect of Bax overexpression on cell proliferation and sensitivity to chemotherapeutic agents in two gastric cancer cells using a CrelloxP-mediated expression system. Methods: We transfected bax-ainto MKN-28 and MKN-45 under the control of an inducible CrelloxP expression system, and stably transfected cells were obtained. Bax expression was induced by infection of a Cre expressing adenoviral vector. Cell viability after induction of bax was measured by the MTT assay. We also examined the cell viability of MKN-28, pre- and post-induction of bax, under some chemotherapeutic agents, and calculated the concentration of each drug yielding 50% inhibition of cell growth. Results: After induction of bax, both cells showed decreased growth rate within 2 days (in MKN28) and 3 days (in MKN-45), compared with infecting LacZ-expressing adenoviral vector or a mock infection, partially due to increased apoptosis. This death-promoting effect of bax was also shown by transient cotransfection assay, a bax-expression plasmid or empty vector with a lacZ expression plasmid into parental MKN-45 cells. The number of lacZpositive cells were markedly reduced in bax-transfected cells (0.2:':0.3% vs. 12.2:':2.1 %,p
Insulin like growth factors (IGFs) are mitogenic for normal and neoplastic cells. Plasma IGF-I levels correlate with the risk of prostate, lung and breast cancer. Acromegalies have high levels ofIGF-I and growth hormone (GH), and show at increased risk of colorectal cancer. We examined the relationship between plasma IGF-I, IGF-ll, IGFBP-3, crypt cell proliferation and apoptosis in an ongoing study of colorectal adenoma risk factors. Methods: Blood and biopsy samples of normal colon were obtained from consenting adult subjects referred to the University of North Carolina Hospitals for colonoscopy. Plasma IGFs and IGFBP-3 were measured by ELISA. Proliferation and apoptosis were measured by whole crypt mitotic counts morphologyrrUNEL respectively. Results: 582 subjects (203 adenomas cases; 35% and 379 none adenoma controls; 65%) were assessed. Comparing cases to controls, there were no statistically significant differences in mean plasma IGF-I, IGF-II and IGFBP-3 levels, and no significant differences in proliferation. Cases had lower apoptosis compared to the controls (p< 0.(01). Consistent with prior studies cases also had significantly higher waist/hip ratio, were more likely to be men and were older. We therefore used logistic regression to examine the association between adenoma status and IGF-I, IGF-II and IGFBP-3 while controlling for age, gender, body mass index (BMI) and smoking. In the multivariate models, only age and gender were significant predictors of adenoma status. Compared to the lowest quartile of IGF-I, those in the highest quartile were not more likely to have an adenoma (adjusted odds ratio [OR] 0.80, 95% CI 0.36-1.76). Similarly, those in the highest quartile of IGF-II were not more likely to have adenomas (OR 0.66, 95% CI 0.26-1.70). Among the cases, insulin, IGF-I, female sex and higher waist/hip ratio were associated with increased proliferation. Only BMI was associated with increased proliferation among the controls. Among all subjects being a case, having elevated IGF-I, IGF-II levels, high waist/hip ratio and female sex were associated with low apoptosis. Elevated levels of IGFBP-3 were associated with increased apoptosis among all the subjects. Conclusions: Plasma IGF-I, IGF-II and IGFBP-3, and proliferation did not correlate with the presence of adenomas. Low levels of apoptosis in the normal colonic mucosa were associated with adenomas indicating that this could be a predictor of adenoma risk.
2830
2832
PROLONGED ACTIVATION OF MITOGEN·ACTIVATED PRO· TEIN KINASES (MAPKS) IN NSAIDS·INDUCED APOPTOSIS. Won Ho Kim, Soo Hyun Jin, Eun Hyae Kang, Tae II Kim, Jin Kyung Kang, Yonsei Univ Coli of Medicine, Seoul, South Korea.
INHIBITION OF APOPTOSIS BY GROWTH FACTORS AND PROSTAGLANDIN· A KEY EVENT IN HEALING OF STRESS DAMAGE? Peter Ch Konturek, Pierzchalski Piotr, Tomasz Brzozowski, Alexandra Szlachcic, Stanislaw J. Konturek, Eckhart G. Hahn, Dept Med I , Univ Med, Erlangen, Germany; Dept Physiol , Univ Med Sch, Cracow, Poland. Growth factors such as epidermal growth factor (EGF) and pancreatic trypsin inhibitor (PSTI) are involved in healing of gastric lesions induced by stress but their influence on apoptosis in the gastric mucosa exposed to stress remains unknown. In this study we compared the ulcer healing effects of EGF and PSTI (50 ILglkgi.p.) with those exhibited by 16,16 dm PGE 2 (5 ILglkgi.p.), and we determined the alterations in apoptosis, gastric blood flow (GBF) and expression of cytokines in rats exposed to 3.5 h of water immersion and restraint stress (WRS). The animals were killed immediately (time 0) and at 3, 6, 12,24 and 48 h after the end ofWRS and the area of gastric lesions was measured by planimetry and gastric blood flow (GBF) was determined by H2-gas clearance method. Blood was collected for measurement of plasma IL-l{3 and TNFa levels by ELISA and mucosal biopsy samples were taken for determination of IL-l {3 and TNFa mRNA and expression of apoptosis-releted proteins such as Bax and Bcl-2 mRNA by RT-PCR and Southern blot. The rate of apoptosis was measured by transferase-mediated dUPT-biotin nick end-labeling (TUNEL) method. After 3.5 h of exposure to WRS gastric erosions occurred and their number reached the maximum at 12 and then declined at 24 h to disappear at 48 h. The GBF was significantly decreased immediately after WRS but then it rose progressively starting from 3 up to 48 h after WRS. Expression of IL-l{3 and TNFa mRNA rose up to 12 h then declined at 24 h and returned to the control level at 48 h after WRS and this was accompanied by the rise in plasma IL-I{3 and TNFa levels. Bax and Bcl-2 mRNA were undetectable in the intact gastric mucosa but at 6 h after WRS, a significant rise in the number of apoptotic cells (TUNEL method) and an imbalance between Bax and Bcl-2 (BaxlBcl-2 ratioz l) were observed. Bax mRNA gradually increased in first 4 hours to decline at 24 h after WRS while Bcl-2 was detected at 6 and 12 h to decline to the control level at 24 h after WRS. Treatment with EGF, PSTI and PGE 2 accelerated significantly healing of WRS lesions, counteracted the imbalance between BaxIBcl-2 proteins and attenuated the rise in apoptosis and plasma IL-l {3 and TNFa levels after WRS. We conclude that: l)stress lesions involve overexpression of inflammatory cytokines, the fall in the GBF and enhancement in apoptosis triggered by the shift from cell death effector Bax to the cell death repressor Bcl-2 and 2)growth factors and PG may contribute to the recovery from WRS lesions by the attenuation of apoptosis and suppression of cytokine expression and release.
Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of and mortality from colon cancer. The mechanisms of the anti-neoplastic effect of NSAIDs are still far from complete understanding, but one of the possible mechanisms is an induction of apoptosis. We have previously reported the role of caspase-3, a key enzyme for the execution of apoptosis, in NSAIDs-induced apoptosis of colon cancer cells. Mitogenactivated protein kinases (MAPKs), such as extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinase (1NK)/stress-activated protein kinase (SAPK) and p38 kinase are activated in response to different stimuli, have different downstream targets and, therefore, perform different functions. Of these MAPKs, JNKlSAPK and p38K are considered as important mediators of apoptotic signals in many systems. Aim: The aim of this study was to demonstrate the activation of MAPKs in NSAIDsinduced apoptosis of colon cancer cells and to elucidate a pathway leading to NSAIDs-induced apoptosis. Subjects and Methods: Apoptosis was detected and confirmed by confocal microscopic observation of chromatin condensation after DAPI stain and the demonstration of DNA fragmentation in agarose gel electrophoresis. Cell death was measuredd by trypan blue dye exclusion method. Caspase-3 activity was measured using tetrapeptide fluorogenic substrate, Ac-DEVD-AMC. MAPKs activation was assessed by Western blot using phospho-specific antibodies to MAPKs. Kinase assay using ATF-2 fusion protein as a substrate was also performed for the measurement of p38K activity. For the inhibition of p38K, pyridinylimidazole compound, SB203580, was utilized. Results: In the colon cancer cell line, HT-29, NSAIDs such as indomethacin, sulindac and nabumetone, induced dose- and time-dependent apoptosis as well as prolonged activation of MAPKs including ERK, JNK and p38K. SB203580, a p38K inhibitor, attenuated not only indomethacin-induced cell death but also caspase-3 activation. However, caspase inhibitors, such as Ac-DEVDCHO, Ac-YVAD-CHO and Z-VAD-FMK, did not affect p38K activation in indomethacin-treated HT-29 cells. Conclusion: From these results, we concluded that prolonged activation of MAPKs including p38K, followed by caspase-3 activation, is an important signal pathway mediating NSAIDs-induced colon cancer cell apoptosis.