Blockade of cholecystokinin-2 receptor and cyclooxygenase-2 synergistically induces cell apoptosis, and inhibits the proliferation of human gastric cancer cells in vitro

Gastrin and cyclooxygenase-2 (COX-2) play important roles in the carcinogenesis and progression of gastric cancer. However, it remains unknown whether the combination of cholecystokinin-2 (CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic anti-tumor effects on human gastric cancer. Here, we demonstrated that the combination of AG-041R (a CCK-2 receptor antagonist) plus NS-398 (a selective COX-2 inhibitor) treatment had synergistic effects on proliferation inhibition, apoptosis induction, down-regulation of Bcl-2 and up-regulation of Bax expression in MKN-45 cells. These results indicate that simultaneous targeting of CCK-2 receptor and COX-2 may inhibit gastric cancer development more effectively than targeting either molecule alone.     

Arsenic trioxide inhibits proliferation and induces apoptosis in pancreatic cancer cells

Because of the poor therapeutic responsiveness of pancreatic cancer patients, new chemotherapeutic agents for pancreatic cancer would be extremely beneficial. The effects of arsenic trioxide in pancreatic cancer have not been explored. To evaluate the anti-pancreatic cancer effects of arsenic trioxide, three human pancreatic cell lines, HPAF, MiaPaCa-2 and PANC-1, were tested. Arsenic trioxide caused dose- and time dependent inhibition of pancreatic cancer cell proliferation. In parallel with inhibition of cell proliferation, arsenic trioxide induced significant morphological changes, including shrunken cytoplasm, membrane blebbing, and nuclear condensation consistent with apoptosis. Propidium iodide DNA staining showed an increase of the sub-G0/G1 cell population. The DNA fragmentation induced by arsenic trioxide in these three cell lines was confirmed by the TUNEL assay. Furthermore, Western blotting analysis indicated that caspase-3 was activated following arsenic trioxide as measured by procaspase-3 cleavage and PARP cleavage. These findings show that arsenic trioxide has potent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.     

Blockade of cholecystokinin-2 receptor and cyclooxygenase-2 synergistically induces cell apoptosis,and inhibits the proliferation of human gastric cancer cells in vitro

Gastrin and cyclooxygenase-2 (COX-2) play important roles in the carcinogenesis and progression of gastric cancer. However, it remains unknown whether the combination of cholecystokinin-2 (CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic anti-tumor effects on human gastric cancer. Here, we demonstrated that the combination of AG-041R (a CCK-2 receptor antagonist) plus NS-398 (a selective COX-2 inhibitor) treatment had synergistic effects on proliferation inhibition, apoptosis induction, down-regulation of Bcl-2 and up-regulation of Bax expression in MKN-45 cells. These results indicate that simultaneous targeting of CCK-2 receptor and COX-2 may inhibit gastric cancer development more effectively than targeting either molecule alone.     

Celecoxib通过环氧合酶2和前列腺素E_2途径抑制胰腺癌JF-305细胞增殖

目的　研究Celecoxib对COX 2高表达人胰腺癌细胞JF 30 5生长和凋亡的影响及作用机制。方法　采用四唑氮蓝 (MTT)比色法检测细胞增殖 ,流式细胞仪测定细胞周期和凋亡 ,酶联免疫吸附试验 (ELISA)检测前列腺素E2 (PGE2 )含量。结果　Celecoxib可明显抑制JF 30 5细胞增殖和诱导其凋亡 ,并且这种增殖抑制作用能被PGE2 拮抗。结论　Celecoxib通过COX 2和PGE2 途径抑制JF 30 5细胞生长和诱导其凋亡 ;Celecoxib可能是COX 2高表达胰腺肿瘤的一种有效的化学治疗和化学预防药物。     

Ganoderma lucidum inhibits proliferation and induces apoptosis in human prostate cancer cells PC-3

Ganoderma lucidum (Reishi), an oriental medical mushroom, has been widely used in Asian countries for centuries to prevent or treat different diseases, including cancer. However, the mechanism(s) responsible for the effects of Ganoderma lucidum on cancer cells remain to be elucidated. We have previously demonstrated that Ganoderma lucidum down-regulated the expression of NF-kappaB-regulated urokinase plasminogen activator (uPA) and uPA receptor (uPAR), which resulted in suppression of cell migration of highly invasive human breast and prostate cancer cells. In this study, we investigated the effects of Ganoderma lucidum on cell proliferation, cell cycle, and apoptosis in human prostate cancer cells PC-3. Our data demonstrate that Ganoderma lucidum inhibits cell proliferation in a dose- and time-dependent manner by the down-regulation of expression of cyclin B and Cdc2 and by the up-regulation of p21 expression. The inhibition of cell growth was also demonstrated by cell cycle arrest at G2/M phase. Furthermore, Ganoderma lucidum induced apoptosis of PC-3 cells with a slight decrease in the expression of NF-kappaB-regulated Bcl-2 and Bcl-xl. However, the expression of proapoptotic Bax protein was markedly up-regulated, resulting in the enhancement of the ratio of Bax/Bcl-2 and Bax/Bcl-xl. Thus, Ganoderma lucidum exerts its effect on cancer cells by multiple mechanisms and may have potential therapeutic use for the prevention and treatment of cancer.     

Wikyungtang inhibits proliferation of A549 human lung cancer cells via inducing apoptosis and suppressing cyclooxygenase-2 activity

Wikyungtang, an oriental herbal formulation, has been known to exert anti-inflammatory and anti-tumoral activity. However, its molecular mechanism of action is not understood. The purpose of the present study was to examine the effect of the water extract of Wikyungtang (WKT) on the growth of A549 human lung cancer cells. Treatment with WKT resulted in a dose-dependent growth inhibition coupled with the characteristic morphological features of apoptosis. Apoptosis-inducing concentrations of WKT induced caspase-3 and caspase-9 activation accompanied by proteolytic degradation of poly(ADP-ribose)-polymerase and phospholipase C-gamma1. In addition, WKT-induced apoptosis in A549 cells was associated with a decreased expression of the anti-apototic Bcl-XL expression. WKT treatment also inhibited the expression of cyclooxygenase (COX)-2 and the accumulation of prostaglandin E2 without significant changes in the levels of COX-1. Taken together, these findings provide important new insights into the possible molecular mechanisms of the anti-cancer activity of WKT.     

Celecoxib通过环氧合酶2和前列腺素E_2途径抑制胰腺癌JF-305细胞增殖

目的　研究Celecoxib对COX 2高表达人胰腺癌细胞JF 30 5生长和凋亡的影响及作用机制。方法　采用四唑氮蓝 (MTT)比色法检测细胞增殖 ,流式细胞仪测定细胞周期和凋亡 ,酶联免疫吸附试验 (ELISA)检测前列腺素E2 (PGE2 )含量。结果　Celecoxib可明显抑制JF 30 5细胞增殖和诱导其凋亡 ,并且这种增殖抑制作用能被PGE2 拮抗。结论　Celecoxib通过COX 2和PGE2 途径抑制JF 30 5细胞生长和诱导其凋亡 ;Celecoxib可能是COX 2高表达胰腺肿瘤的一种有效的化学治疗和化学预防药物。     

The cyclooxygenase-2 inhibitor NS-398 inhibits proliferation and induces apoptosis in human osteosarcoma cells via downregulation of the survivin pathway

Cyclooxygenase-2 (COX-2) is frequently overexpressed in human malignancies and plays a crucial role in tumorigenesis and cancer progression. The present study aimed to investigate the expression and clinical significance of COX-2 and survivin (SUV) in human osteosarcomas (OS), and explore the effects and molecular mechanisms of a selective COX-2 inhibitor NS-398 and SUV on tumor proliferation and apoptosis. Fifty cases of human OS and osteochondromas (OC) were collected. The expression of COX-2 and SUV was assessed using immunohistochemical assays in biopsy samples. MG-63 human OS cells were treated with different concentrations of NS-398, used to investigate their effects on cell proliferation and apoptosis. The recombinant small hairpin RNA adenovirus vector rAd5-SUV was constructed, and the effects and molecular mechanisms of knockdown of SUV on proliferation and apoptosis were evaluated in MG-63 cells. A subcutaneous xenograft tumor model was established, validating the effects of rAd5-SUV on tumor growth in vivo. Based on the results, the expression of COX-2 and SUV in OS showed a higher strong reactivity rate compared with OC (73.3 vs. 25.0%, P=0.001; 63.3 vs. 30.0%, P=0.02), but it did not correlate with the clinicopathological characteristics of OS. NS-398 inhibited proliferation, induced apoptosis and decreased the mRNA expression of COX-2 and SUV in MG-63 cells. Furthermore, adenovirus-mediated knockdown of SUV inhibited proliferation, induced apoptosis, reduced the expression of proliferating cell nuclear antigen (PCNA), increased the expression of caspase-3 (CAS-3) and slowed the growth of xenograft tumors in MG-63 cells. Taken together, the expression of COX-2 and SUV is closely correlated with human OS, and inhibition of COX-2 or knockdown of SUV suppresses tumor proliferation and induces apoptosis, suggesting that COX-2 may be involved in OS cell proliferation and apoptosis through SUV-mediated regulation of PCNA and CAS-3 expression, and provides a potential therapeutic strategy for the treatment of cancer.     

Actinomycin D induces apoptosis and inhibits growth of pancreatic cancer cells

Pancreatic cancer cells are usually resistant to apoptosis induced by cytotoxic drugs, by activation of surface receptors such as Fas and TNF receptor or by serum or growth factor withdrawal. Actinomycin D (actD) is an inhibitor of RNA synthesis and acts as a potent inducer of apoptosis in several cell lines. In the present study, we investigated the effects of actD on PANC-1 pancreatic cancer cells. ActD caused apoptosis in PANC-1 cells in a dose-dependent manner, as determined by cell growth assays, DNA laddering and TUNEL assays. Induction of apoptosis correlated with activation of the JNK/SAPK pathway and increased expression of Bax but not Bad or p53. PANC-1 cells were completely resistant to Fas antibody and TNF-alpha. In contrast, TRAIL decreased the growth of PANC-1 cells by 22%. Low concentrations of actD (10 ng/ml) enhanced the cytotoxic effects of all 3 cytokines. EGF, FGF-2 and IGF-I did not protect PANC-1 cells from actD-mediated apoptosis. ActD (10 ng/ml) also inhibited the growth of CAPAN-1 and T3M4 pancreatic cancer cells but not MiaPaCa-2 cells. Our observations suggest that actD may act via JNK/SAPK and Bax to promote apoptosis in PANC-1 cells and that it may inhibit the growth of other pancreatic cancer cell lines.     

Ciprofloxacin induces apoptosis and inhibits proliferation of human colorectal carcinoma cells

Efficacy of chemotherapy in advanced stages of colorectal tumours is limited. The quinolone antibiotic ciprofloxacin was recently shown to inhibit growth and to induce apoptosis in human bladder carcinomas cells. We investigated the effect of ciprofloxacin on colon carcinoma lines in vitro. CC-531, SW-403 and HT-29 colon carcinoma and HepG2 hepatoma cells (control cells) were exposed to ciprofloxacin. Proliferation was assessed by bromodeoxyuridine-incorporation into DNA and apoptosis was measured by flow cytometry after propidium iodide or JC-1 staining. Expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax was analyzed by semiquantitative Western blot analysis and activity of caspases 3, 8 and 9 by substrate-cleavage assays. Ciprofloxacin suppressed DNA synthesis of all colon carcinoma cells time- and dose-dependently, whereas the hepatoma cells remained unaffected. Apoptosis reached its maximum between 200 and 500 microg ml(-1). This was accompanied by an upregulation of Bax and of the activity of caspases 3, 8 and 9, and paralleled by a decrease of the mitochondrial membrane potential. Ciprofloxacin decreases proliferation and induces apoptosis of colon carcinoma cells, possibly in part by blocking mitochondrial DNA synthesis. Therefore, qualification of ciprofloxacin as adjunctive agent for colorectal cancer should be evaluated.     

Doxycycline induces caspase-dependent apoptosis in human pancreatic cancer cells

Doxycycline (DC) belongs to the tetracycline family of antibiotics and has been used clinically for over 5 decades. Despite advances in understanding the molecular pathogenesis of pancreatic cancer, no chemotherapy course has shown significant effectiveness. Hence new treatments are needed. In this study we report the pro-apoptotic effects of DC in 2 pancreatic adenocarcinoma cell lines, T3M4 and GER. Cell proliferation was measured using the SRB protein dye. Induction of apoptosis was detected using ELISA. Caspase activation was detected using either immunoblotting or a colorimetric assay based on cleavage of caspase-associated substrates. Expression of proteins and post-translational modifications were determined using immunoblotting. Treatment of pancreatic cancer cells with DC reduces their proliferation. This reduction is, at least partly, due to increased caspase-dependent apoptosis involving activation of caspase3, caspase7, caspase8, caspase9, caspase10 and increased levels of FADD. Inhibition of caspase8 or caspase10 but not caspase9 significantly decreases DC-induced apoptosis in both cell lines. Furthermore treatment of pancreatic cancer cells with DC increases protein levels of Bax and phosphorylation of members of the p38MAPK pathway such as p38MAPK, MKK3/6 and MAPKAPK2. These results provide an insight into mechanisms behind the pro-apoptotic effects of DC in pancreatic cancer cells.     

Parthenolide induces proliferation inhibition and apoptosis of pancreatic cancer cells in vitro

Background

To explore the anti-tumor effects of parthenolide in human pancreatic cancer.

Methods

BxPC-3 cell, a human pancreatic cancer, was treated with parthenolide at different concentrations. The MTT assay was used to analyze cell viability. Flow cytometry and DNA fragmentation analysis were applied to evaluate apoptosis after parthenolide treatment. The wound closure and cell invasion assay were also employed in the study. Western blotting was used to demonstrate Bad, Bcl-2, Bax, caspase-9 and pro-caspase-3 expression.

Results

The MTT assay indicated that the pancreatic cancer growth could be dose-dependently inhibited by parthenoolide. This phenomenon was confirmed by flow cytometry and DNA fragmentation analysis. The wound closure assay and cell invasion assay showed that BxPC-3 cell was significantly suppressed by parthenolide at 7.5 μM and 15 μM. Western Blotting demonstrated the Bcl-2 and pro-caspase-3 were down-regulated while the Bax and caspase-9 were up-regulated. No alteration in Bad expression was found after treatment.

Conclusions

The parthenolide can inhibit the cell growth, migration, and induce the apoptosis in human pancreatic cancer. These findings may provide a novel approach for pancreatic cancer treatment.     

Triptolide inhibits proliferation and induces apoptosis of human melanoma A375 cells

Triptolide, a diterpenoid obtained from Tripteryglum wilfordii Hook.f, has attracted interest for its anti- tumor activities against human tumor cell lines in recent years. This report focuses on anti-proliferative and pro-apoptotic activities in human melanoma A375 cells assessed by CCK8 assay, Hoechst 33258 staining and flow cytometry. In addition, triptolide-induced arrest in the S phase was also observed. Caspase assays showed the apoptosis induced by triptolide was caspase-dependent and probably through intrinsic apoptotic pathways. Furthermore, expression of NF-κB (p65) and its downstream factors such as Bcl-2, Bcl-XL was down-regulated. Taken together, the data indicate that triptolide inhibits A375 cells proliferation and induces apoptosis by a caspase-dependent pathway and through a NF-κB-mediated mechanism.     

藤黄酸抑制前列腺癌PC-3细胞增殖并诱导其细胞凋亡

目的:研究中药藤黄的有效成分藤黄酸(gambogic acid,GA)对前列腺癌PC-3细胞增殖和凋亡的影响。方法:采用不同浓度的GA作用前列腺癌PC-3细胞后,在体外通过CCK-8比色法分析细胞的增殖情况,吖啶橙/溴化乙啶(acridine orange/ethidium bromide,AO/EB)双重染色法和FCM法分析细胞的凋亡情况;蛋白质印迹法检测细胞中凋亡相关蛋白P53、Bax和Bcl-2的表达变化。结果:GA不仅能抑制PC-3细胞的增殖,而且能有效诱导其细胞凋亡,与对照组比较差异有统计学意义(P<0.05),并且其抑制增殖和促凋亡作用呈浓度依赖性。CCK-8法检测结果表明,GA浓度>1μmol/L时,细胞的增殖能力受到明显抑制。AO/EB染色法显示,GA处理后的前列腺癌PC-3细胞核呈致密浓染橘红色,并伴有核浓缩和偏向。FCM法检测结果显示,GA处理后的前列腺癌PC-3细胞凋亡峰明显。蛋白质印迹法进一步表明,GA能够上调PC-3细胞中Bax和P53的表达水平,下调Bcl-2表达水平。结论:GA对前列腺癌PC-3细胞具有明显的抑制增殖和诱导凋亡作用。     

Protease-activated receptor-2 regulates cell proliferation and enhances cyclooxygenase-2 mRNA expression in human pancreatic cancer cells

BACKGROUND AND OBJECTIVES: Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor that is activated by trypsin. Recent studies have suggested that PAR-2 activity correlates with inflammatory processes and cell proliferation and that PAR-2 activation in non-neoplastic cells induces expression of cyclooxygenase-2 (COX-2). In the present study, we examined whether PAR-2 activation regulates cell proliferation and COX-2 expression by pancreatic cancer cells. METHODS: We analyzed PAR-2 expression immunohistochemically in 40 intraductal papillary-mucinous neoplasms (IPMNs) and 73 invasive ductal carcinomas (IDCs) of the pancreas. We used four pancreatic cancer cell lines (Panc1, T3M4, BxPC3, and MIApaca2) to measure cell proliferation and COX-2 mRNA expression after PAR-2 activation. RESULTS: PAR-2 protein was detected immunohistochemically in 85.0% of IPMNs and 65.8% of IDCs. Trypsin and a PAR-2 agonist peptide, SLIGKV, stimulated proliferation of each cell line in a dose-dependent manner. Exposure of cells to anti-PAR-2 neutralizing antibody prior to PAR-2 activation suppressed cell proliferation. In COX-2-positive cell lines (T3M4 and BxPC3), PAR-2 activation significantly increased COX-2 mRNA expression. CONCLUSIONS: Our results suggest that PAR-2 activation is associated with cell proliferation and COX-2 expression in pancreatic cancer cells. Blockade of the PAR-2 signaling pathway may be a novel strategy for suppressing pancreatic tumor growth.     

Cyclin B1 depletion inhibits proliferation and induces apoptosis in human tumor cells

Cyclin B1 is the regulatory subunit of M-phase promoting factor, and proper regulation of cyclin B1 is essential for the initiation of mitosis. Increasing evidence indicates that the deregulation of cyclin B1 is involved in neoplastic transformation, suggesting the suppression of cyclin B1 could be an attractive strategy for antiproliferative therapy. In the present work, we analysed the impact of small interfering RNAs (siRNAs) targeted to cyclin B1 on different human tumor cell lines. Cyclin B1 siRNAs reduced the protein level of cyclin B1 in HeLa, MCF-7, BT-474 and MDA-MB-435 tumor cells and efficiently reduced the kinase activity of Cdc2/cyclin B1 in HeLa cells. siRNA-treated cells were arrested in G2/M phase in all tumor cell lines tested. Proliferation of tumor cells from different origins was suppressed by 50-80% 48 h after transfection and apoptosis was increased from 5 to 40-50%. Furthermore, tumor cells showed less colony-forming ability after siRNA treatment. In contrast, primary human umbilical vein endothelial cells exhibited only a slight change in cell cycle, and neither apoptosis nor clear inhibition of proliferation was observed after cyclin B1 siRNA treatment for 48 h. These results indicate that siRNAs against cyclin B1 could become a powerful antiproliferative tool in future antitumor therapy.     

FTY720, a synthetic compound from Isaria sinclairii, inhibits proliferation and induces apoptosis in pancreatic cancer cells

FTY720, a synthetic compound produced by modification of a metabolite from Isaria sinclairii, is known as a unique immunosuppressive agent that exerts its activity by inducing apoptosis of lymphocytes [S. Suzuki, FTY720: Mechanisms of action and its effect on organ transplantation, Transplant. Proc. 31 (1999) 2779-2782]. Additionally, it has been found that FTY720 has inhibitory effects on various cancer growth and metastasis [J.D. Wang, S. Takahara, N. Nonomura, Early induction of apoptosis in androgen-independent prostate cancer cell line by FTY720 requires caspase-3 activation, Prostate 40 (1999) 50-55]. To investigate its effect on the growth and metastasis of pancreatic cancer, FTY720 was used to treat three pancreatic cancer cell lines (BxPC-3, AsPC-1, and PANC-1). The MTT assay and flow cytometry were used to evaluate the cell death after FTY720 treatment; the wound closure assay, three-dimensional (3D) Matrigel assay, and invasive assay were used to evaluate the migration, colony formation and invasion abilities after FTY720 treatment, respectively. Protein expression in BxPC-3, AsPC-1, and PANC-1 cells after FTY720 treatment was detected by Western blotting. The MTT assay indicated that the growth of pancreatic cancer cells could be inhibited by FTY720 at various concentrations between 0 and 17 microM in a dose-dependent manner, which was also confirmed by flow cytometry. The wound closure assay, 3D Matrigel assay and cell invasion assay all showed that FTY720 significantly suppressed migration, colony formation and invasion ability of cancer cells at concentrations from 5 to 17 microM. After FTY720 treatment, the phospho-Akt, Bcl-2, pro-caspase-3 expression were down-regulated while the caspase-9 protein expression was increased. In conclusion, FTY720 can inhibit the growth, migration and invasion of pancreatic cancer cells. Our study provides a preclinical support for chemotherapeutic approach with FTY720 for the treatment of pancreatic cancer.     

Resveratrol inhibits cell proliferation and induces apoptosis of human breast carcinoma MCF-7 cells

Resveratrol, which is found in grapes and wine, has been reported to have a variety of important pharmacological effects including anti-inflammatory, anti-platelet, and anti-carcinogenetic properties. In this study, using the human breast cancer cell line MCF-7, we have analyzed a possible mechanism by which resveratrol could interfere with cell cycle control and induce cell death. Resveratrol treatment of MCF-7 cells resulted in a dose-dependent inhibition of the cell growth and the cells accumulated at the S phase transition of the cell cycle at low concentrations, but high concentrations do not induce S phase accumulation. The anti-proliferative effects of resveratrol were associated with a marked inhibition of cyclin D and cyclin-dependent kinase (Cdk) 4 proteins, and induction of p53 and Cdk inhibitor p21WAF1/CIP. Growth suppression by resveratrol was also due to apoptosis, as seen by the appearance of a sub-G1 fraction and chromatin condensation. In addition, the apoptotic process involves activation of caspase-9, a decrease of Bcl-2 as well as Bcl-XL levels, and an increase of Bax levels.