Cyclooxygenase-2 inhibitor, nimesulide, improves radiation treatment against non-small cell lung cancer both in vitro and in vivo

Lung cancer is the leading cause of cancer-related deaths in the United States. Despite improvements in radiation, surgery and chemotherapy the 5 year survival statistics of non-small cell lung cancer (NSCLC) have improved little over the past two decades. It has been proposed that NF-kappaB is a participant in the cytoprotection against several redox-mediated therapeutic agents including ionizing radiation. Cyclooxygenase-2 (COX-2) inhibition has become an attractive target for enhancing the efficacy of radiation and chemotherapy. Numerous mechanistic pathways have been proposed as the means through which COX-2 inhibition enhances the efficacy of radiation. We hypothesize that the COX-2 inhibitor, nimesulide, will improve the efficacy of radiation therapy (RT), at least in part, via the suppression of NF-kappaB mediated cytoprotective pathways. In this study we used the COX-2 inhibitor nimesulide to improve the efficacy of RT when measured by tumor regrowth assays in vivo and clonegenic survival in vitro. For the in vivo assay, A549 tumor cells representing NSCLC were subcutaneously injected into the right flanks of female athymic nude mice (n=10/group). Mice were given nimesulide via drinking water at a concentration of 5 microg/g body weight (b.w.) and the water was replenished daily. Tumors were treated with 30 Gy fractionated radiation and measured bi-weekly. For our in vitro study, clonogenic survival assays were evaluated to determine the effect of nimesulide, radiation, and the combination. The NF-kappaB mediated mechanism of nimesulide was measured by Western blot analysis of NF-kappaB target genes, MnSOD and survivin. In vivo, mice that received combined treatments of 5 microg/g b.w. nimesulide and 30 Gy radiation (3 Gy/fraction, 10 daily fractions) had significant reduction in tumor size in comparison to the 30 Gy radiation control group (p<0.05). In vitro, nimesulide alone produced a significant decrease in clonogenic survival at doses from 0-300 microM. Nimesulide demonstrated an additive effect in combination with radiation. Nimesulide alone reduced MnSOD and survivin protein levels in a dose-dependent manner. 6 Gy radiation caused an initial elevation of MnSOD protein levels which was inhibited by prior treatment of nimesulide suggesting an inhibition of radiation induced NF-kappaB target genes. These results support the hypothesis that COX-2 inhibitors such as nimesulide can increase the efficacy of radiation therapy. In vitro, our results suggest that the radiosensitization of A549 tumor cells by nimesulide is mediated by the suppression of NF-kappaB-mediated, radiation-induced cytoprotective genes.     

Celecoxib and NS-398 enhance photodynamic therapy by increasing in vitro apoptosis and decreasing in vivo inflammatory and angiogenic factors

Photodynamic therapy (PDT) elicits both apoptotic and necrotic responses within treated tumors and produces microvascular injury leading to inflammation and hypoxia. PDT also induces expression of angiogenic and survival molecules including vascular endothelial growth factor, cyclooxygenase-2 (COX-2), and matrix metalloproteinases. Adjunctive administration of inhibitors to these molecules improves PDT responsiveness. In the current study, we examined how the combination of PDT and COX-2 inhibitors improve treatment responsiveness. Photofrin-mediated PDT combined with either celecoxib or NS-398 increased cytotoxicity and apoptosis in mouse BA mammary carcinoma cells. Immunoblot analysis of protein extracts from PDT-treated cells also showed poly(ADP-ribose) polymerase cleavage and Bcl-2 degradation, which were further enhanced following combined therapy. Tumor-bearing mice treated with PDT and either celecoxib or NS-398 exhibited significant improvement in long-term tumor-free survival when compared with PDT or COX-2 inhibitor treatments alone. The combined procedures did not increase in vivo tumor-associated apoptosis. Administration of celecoxib or NS-398 attenuated tissue levels of prostaglandin E2 and vascular endothelial growth factor induced by PDT in treated tumors and also decreased the expression of proinflammatory mediators interleukin-1beta and tumor necrosis factor-alpha. Increased tumor levels of the antiinflammatory cytokine, interleukin 10, were also observed following combined treatment. This study documents for the first time that adjunctive use of celecoxib enhances PDT-mediated tumoricidal action in an in vivo tumor model. Our results also show that administration of COX-2 inhibitors enhance in vitro photosensitization by increasing apoptosis and improve in vivo PDT responsiveness by decreasing expression of angiogenic and inflammatory molecules.     

Enhancement of photodynamic therapy by 2,5-dimethyl celecoxib, a non-cyclooxygenase-2 inhibitor analog of celecoxib

Photodynamic therapy (PDT) effectiveness can be improved by employing combined modality approaches involving pharmaceuticals targeting the tumor microenvironment and/or tumor cell death pathways. In one approach, combining PDT with celecoxib improves long-term tumoricidal activity without increasing normal tissue photosensitization. However, side effects arising from the use of coxib based cyclooxygenase-2 (COX-2) inhibitors, including cardiovascular injury, decreases the clinical applications of this class of compounds. A growing number of studies demonstrate that the tumoricidal actions of coxibs such as celecoxib involve non-COX-2 mediated mechanisms. The celecoxib analog, 2,5-dimethyl celecoxib (DMC), lacks COX-2 inhibitory activity but exhibits cytotoxic properties comparable to the COX-2 inhibitor celecoxib. We compared the effectiveness of DMC and celecoxib in modulating PDT response at both the in vitro and in vivo level using a C3H/BA murine mammary carcinoma model. Both DMC and celecoxib blocked PDT induced expression of the pro-survival protein survivin, enhanced the endoplasmic reticulum stress (ERS) response of PDT, and increased both apoptosis and cytotoxicity in BA cells exposed to combination protocols. DMC enhanced the in vivo tumoricidal responsiveness of PDT without altering PGE2 levels. Our data demonstrates that DMC improved PDT by increasing apoptosis and tumoricidal activity without modulating COX-2 catalytic activity. Our results also suggest that celecoxib mediated enhancement of PDT may involve both COX-2 dependent and independent mechanisms.     

Effect of a selective cyclooxygenase-2 inhibitor, nimesulide, on the growth of lung tumors and their expression of cyclooxygenase-2 and peroxisome proliferator- activated receptor-gamma.

PURPOSE: The objectives of this study were to evaluate the effect of a cyclooxygenase (COX)-2 inhibitor, nimesulide, on the growth inhibition of s.c. human lung A549 adenocarcinoma tumors and to assess the effect of nimesulide on the expression of COX-2 and peroxisome proliferator-activated receptor (PPAR)-gamma in lung tumors harvested from mice. EXPERIMENTAL DESIGN: Female nu/nu mice were xenografted with s.c. A549 lung tumors, and 1 day after tumor implantation, the mice were fed with a diet containing nimesulide at 250-1500 ppm doses. Tumor dimensions were monitored twice weekly, and tumor samples isolated from mice were used to determine prostaglandin E(2) (PGE(2)) levels by enzyme immunoassay, expression of COX-2 and PPAR-gamma by Western blotting and immunohistochemistry. Furthermore, the induction of apoptosis in tumor specimens was determined by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. RESULTS: Nimesulide treatment showed a dose-dependent growth-inhibitory effect of A549 tumors with a maximum of 77.7% inhibition at 1500 ppm of nimesulide. Western blotting experiments showed similar expression of COX-2 in both control and nimesulide (250-1500 ppm)-treated mice tumor tissues. PPAR-gamma was found to be overexpressed as a result of 1500 ppm nimesulide treatment and was not detected in tumors from control or 250-1000 ppm nimesulide-treated mice. Nimesulide (1500 ppm) significantly reduced intratumor PGE(2) levels (P < 0.001) and induced apoptosis in 25% of tumor cells as compared with control tumors. CONCLUSIONS: Nimesulide (1500 ppm) induced growth inhibition of A549 lung tumors is associated with the reduction of intratumor PGE(2) levels but without affecting the expression of COX-2. Nimesulide-induced enhancement of the expression of PPAR-gamma may also contribute to its antitumor effect, which needs to be further investigated.     

Cyclooxygenase-2 inhibitor celecoxib augments chemotherapeutic drug-induced apoptosis by enhancing activation of caspase-3 and -9 in prostate cancer cells

Many tumors constitutively express high levels of the inducible form of proinflammatory enzyme, cyclooxygenase-2 (COX-2). Increased COX-2 expression is associated with tumor cell resistance to many cytotoxic chemotherapy drugs. Furthermore, increased resistance to cytotoxic antitumor drugs is also known to be dependent on associated stromal cells in many tumors. We investigated whether prostate tumor-associated stromal cells, marrow-derived osteoblasts, affect cytotoxicity of 2 antitumor drugs, COL-3 and docetaxel (TXTR), and whether it is dependent on COX-2 activity. We further examined whether inhibiting the activity of COX-2 negate the stroma-induced decrease in drug sensitivity in tumor cells. COX-2-specific inhibitor celecoxib (CXB) was used to inhibit COX-2 activity and associated alteration in cell death signaling was investigated. Coculturing PC-3ML cells with osteoblasts decreased the cytotoxicity of the tested antitumor drugs and was associated with increased COX-2 activity in PC-3ML cells. A significant decrease in drug-induced PGE(2) increase and an increase in cytotoxicity were observed when cells were treated with COL-3 or TXTR combined with CXB. Cytotoxicity of single or combination treatment increased apoptosis, which was associated with caspase-3 and -9 activation, PARP cleavage, increased BAD protein, but decreased protein levels of XIAP and BCL-(xL). Oral administration of CXB (40 mg/kg) to mice with PC-3ML tumors for 42 days increased tumor latency, decreased tumor growth and enhanced tumor control with COL-3 or TXTR. Overall, a synergistic enhancement of antitumor activity in combination treatment was observed in vitro and an additive effect in vivo. These observations suggest a potential clinical use of combined dosing of COX-2 inhibitors and cytotoxic drugs at lower, nontoxic dose than currently used to treat advanced prostate cancer.     

Effective photoimmunotherapy of murine colon carcinoma induced by the combination of photodynamic therapy and dendritic cells.

PURPOSE: The unique mechanism of tumor destruction by photodynamic therapy (PDT), resulting from apoptotic and necrotic killing of tumor cells accompanied by local inflammatory reaction and induction of heat shock proteins (HSPs), prompted us to investigate the antitumor effectiveness of the combination of PDT with administration of immature dendritic cells (DCs). EXPERIMENTAL DESIGN: Confocal microscopy and Western blotting were used to investigate the influence of PDT on the induction of apoptosis and expression of HSP expression in C-26 cells. Confocal microscopy and flow cytometry studies were used to examine phagocytosis of PDT-treated C-26 cells by DCs. Secretion of interleukin (IL)-12 was measured with ELISA. Cytotoxic activity of lymph node cells was evaluated in a standard (51)Cr-release assay. The antitumor effectiveness of PDT in combination with administration of DCs was investigated in in vivo model. RESULTS: PDT treatment resulted in the induction of apoptotic and necrotic cell death and expression of HSP27, HSP60, HSP72/73, HSP90, HO-1, and GRP78 in C-26 cells. Immature DCs cocultured with PDT-treated C-26 cells efficiently engulfed killed tumor cells, acquired functional features of maturation, and produced substantial amounts of IL-12. Inoculation of immature DCs into the PDT-treated tumors resulted in effective homing to regional and peripheral lymph nodes and stimulation of cytotoxic activity of T and natural killer cells. The combination treatment with PDT and administration of DCs produced effective antitumor response. CONCLUSIONS: The feasibility and antitumor effectiveness demonstrated in these studies suggest that treatment protocols involving the administration of immature DCs in combination with PDT may have clinical potential.     

Growth stimulation of COX-2-negative pancreatic cancer by a selective COX-2 inhibitor

Cyclooxygenase 2 (COX-2) inhibitors are promising antiangiogenic agents in several preclinical models. The aim of the present study was to evaluate the effect of selective COX-2 inhibitors on vascular endothelial growth factor (VEGF) production in vitro and angiogenesis and growth of pancreatic cancer in vivo, focusing on putative differences between COX-2-negative and COX-2-positive tumors. VEGF production and angiogenesis in vitro were determined by ELISA and endothelial cell migration assay. To determine whether the effect of COX-2 inhibitors was mediated by peroxisome proliferator-activated receptor gamma (PPAR-gamma), we used a dominant-negative PPAR-gamma and a pharmacologic inhibitor. In vitro findings were validated in a pancreatic cancer animal model. Microvessel density was assessed by CD31 immunostaining. Intratumoral prostaglandin and VEGF levels were measured by mass spectroscopy and ELISA. Selective COX-2 inhibitors had a concentration-dependent effect on VEGF production in vitro. Higher concentrations increased VEGF levels and stimulated angiogenesis by activating PPAR-gamma. In vivo, nimesulide increased VEGF production by cancer cells in COX-2-positive and COX-2-negative pancreatic tumors. In COX-2-negative pancreatic cancer, this effect was associated with an increase in angiogenesis and growth. In COX-2-positive pancreatic cancer, the nimesulide-induced increase of VEGF production by the cancer cells was offset by a decrease in VEGF production by the nonmalignant cell types leading to reduced tumor angiogenesis and growth. Selective COX-2 inhibitors had opposite effects on growth and angiogenesis in pancreatic cancer depending on COX-2 expression. These findings imply that assessing the COX-2 profile of the pancreatic tumor is mandatory before initiating therapy with a selective COX-2 inhibitor.     

Inhibition of cytotoxicity of cisplatin by cyclooxygenase-2 inhibitor nimesulide in head and neck cancer cell lines

Although head and neck cancer is a common malignancy, investigations have not yet improved the poor prognosis of patients. Therefore, it is important to find new cancer treatment modalities. Recent studies showed that cyclooxygenase, especially its isoform cyclooxygenase-2, is involved in tumorigenesis, tumor growth and metastasis. Inhibition of this enzyme by cyclooxygenase inhibitors has been shown to be antiproliferative in numerous cancer cell lines. The aim of this study was to investigate if the selective cyclooxygenase-2 inhibitor nimesulide could enhance cytotoxicity of the standard chemotherapeutic agent cisplatin. Head and neck squamous cell cancer cells were incubated with nimesulide and/or cisplatin, and counted after 24, 48 and 72 h treatment. Visualization of apoptotic cells was done by immunohistochemistry. We demonstrated that nimesulide inhibits the cytotoxic effect of cisplatin in HNSCC cells. Therefore, COX-2 inhibitor nimesulide may not be a good partner for cisplatin in combination therapy for cancer.     

Cyclooxygenase-2 inhibitor induces apoptosis and enhances cytotoxicity of various anticancer agents in non-small cell lung cancer cell lines.

In recent years, a combination of two demographic phenomena, an increase in the number of older people in the population and an increase in the incidence of lung cancer with age, has made it mandatory to develop therapeutic modalities with less toxicity for the treatment of inoperable elderly patients with lung cancer. Our study shows that a cyclooxygenase (COX)-2 inhibitor, nimesulide, can inhibit proliferation of non-small cell lung cancer cell lines in vitro in a dose-dependent manner, in part by inducing apoptosis even at clinically achievable low concentrations. Our observations also suggest that the responsiveness of non-small cell lung cancer to COX-2 inhibitors does not require the presence of wild-type p53, but may be influenced by the degree of COX-2 expression. In addition, we found that nimesulide, when used in combination at clinically achievable concentrations, reduced the IC50 values of various anticancer agents by up to 77%, although the level of reduction varied considerably. Because our previous studies have indicated a significantly increased COX-2 expression in up to 70% of adenocarcinoma cases, the present findings are of great clinical interest. In conjunction with the recent development of next generation, highly selective COX-2 inhibitors, they can be expected to lead to even greater efficacy of their use as adjuncts to various anticancer agents for the treatment of high-risk patients without compromising their quality of life.     

Combined effects of cyclooxygenase-1 and cyclooxygenase-2 selective inhibitors on intestinal tumorigenesis in adenomatous polyposis coli gene knockout mice

As with cyclooxygenase (COX)-2, genetic disruption of COX-1 gene or pharmacologic inhibition of its activity has been shown to decrease the number of intestinal polyps in Apc gene-deficient mice. The present study was designed to investigate the combined effects of COX-1 and COX-2 selective inhibitors on spontaneous polyp formation in APC1309 female mice. The animals were treated with 300 or 600 ppm mofezolac (a COX-1 selective inhibitor) alone, 200 or 400 ppm nimesulide (a COX-2 selective inhibitor) alone, 300 ppm mofezolac plus 200 ppm nimesulide, 600 ppm mofezolac plus 400 ppm nimesulide, or 10 ppm indomethacin (a dual-COX inhibitor) in the diet from 7 weeks of age for 4 weeks. Percentage inhibition of polyp area in the intestine was 17% with 600 ppm mofezolac alone and 25% with 400 ppm nimesulide alone, their sum of 42% being almost equal to the 37% observed for the combination treatment. Administration of 300 ppm mofezolac plus 200 ppm nimesulide also significantly decreased polyp area in the intestine by 30%. Moreover, the numbers of polyps more than 2.5 mm in diameter were markedly decreased by combined treatment of both COX inhibitors. With 10 ppm indomethacin, the dual inhibitor, polyp area was also clearly reduced by 46%. Our results indicate that COX-1 and COX-2 may to some extent contribute to polyp formation independently and inhibitor combination treatment thus has particular potential for chemoprevention of colon carcinogenesis.     

Combination of a selective cyclooxygenase-2 inhibitor with epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 and protein kinase A antisense causes cooperative antitumor and antiangiogenic effect.

PURPOSE: Epidermal growth factor receptor (EGFR) and protein kinase A type I(PKAI) play an important role in the control of cancer cell growth and angiogenesis. Inhibitors of EGFR and PKAI have antitumor activity in vitro and in vivo in a variety of tumor types, and some of these agents are active after oral administration. Increasing evidence shows that cyclooxygenase (COX)-2 also plays a role in promoting cancer cell proliferation and angiogenesis. COX-2 expression can be induced by EGFR activation and is regulated by cAMP and PKA. Combination of an EGFR inhibitor with a nonselective COX-1/COX-2 inhibitor prevents the development of intestinal cancer in nude mice. Therefore, we investigated whether any cooperative antitumor effect can be obtained by the combined blockade of COX-2, EGFR, and PKAI. EXPERIMENTAL DESIGN: The COX-2 inhibitor SC-236 was combined with the selective EGFR tyrosine kinase inhibitor ZD1839 (Iressa) and the DNA/RNA-mixed backbone oligonucleotide AS-PKAI to study their effect on human cancer growth and angiogenesis, measuring vascular endothelial growth factor (VEGF) and basic fibroblast growth factor expression and vessel formation, in vitro and after oral administration of these agents in mice. RESULTS: A cooperative effect was observed with SC-236 in combination with either ZD1839 or AS-PKAI, as well as with all three agents together, on the proliferation of human colon and breast cancer cells in soft agar at doses that were ineffective for each agent alone. The antiproliferative effect was accompanied by inhibition of COX-2 expression. Moreover, combination of SC-236 with either agent or the triple combination markedly reduced VEGF secretion in the conditioned medium and completely suppressed VEGF and basic fibroblast growth factor expression. In nude mice bearing human colon cancer xenografts, a low, noninhibitory dose of SC-236 with ZD1839 and AS-PKAI, all given p.o., caused a dramatic cooperative antitumor effect, with no histological evidence of tumor in 60% of mice 5 weeks after treatment withdrawal, at which time all mice were alive. Moreover, analysis of tumor specimens revealed inhibition of vessel formation and expression of COX-2 and VEGF. CONCLUSIONS: This is the first demonstration that three novel agents blocking multiple signaling pathways, in absence of cytotoxic drugs, may have a potent antitumor and antiangiogenic activity after oral administration. Because all agents are under clinical evaluation, our results provide a rationale to translate this feasible therapeutic strategy into a clinical setting.     

High expression of GADD-45alpha and VEGF induced tumor recurrence via upregulation of IL-2 after photodynamic therapy using NPe6

NPe6 is a novel second-generation photosensitizer used for photodynamic therapy (PDT). PDT using NPe6 and diode laser (664 nm) induces cell death, inflammatory reactions, immunological responses and damage to the microvasculature. In this study, we evaluated the influence of the immunological responses and of enhanced angiogenesis on the anti-tumor effect of NPe6-PDT using cytokine-overexpressing Lewis lung carcinoma (LLC), LLC-IL-2 cells both in vitro and in vivo. We showed by DNA microarray analysis in vitro that IL-2 and GADD-45alpha (growth arrest and DNA damage 45 alpha) mRNA expressions were induced by 3 h after NPe6-PDT applied at a dose killing 90% of the cells (LD90). IL-2-overexpressing cells (LLC/IL-2 cells) were resistant to the loss of clonogenicity as compared to the parental LLC cells in vitro. Furthermore, in female C57BL/6 mice, NPe6-PDT produced a cure rate of 66.7% in LLC tumors, whereas the cure rate was only 16.6% in LLC/IL-2 tumors, and overexpression of IL-2 caused failure of NPe6-PDT, with tumor recurrence, in vivo. These results suggest that IL-2 expression may play an unfavorable role in attenuation of the antitumor effect of NPe6-PDT. It has been reported that the expression of vascular endothelial growth factor (VEGF), in particular, may cause tumor recurrence after PDT and exert unfavorable effect in relation to attenuate the anti-tumor activity of PDT. Results of immunohistochemical analysis of LLC/IL-2 tumors have revealed that the expressions of GADD-45alpha and VEGF are induced in these tumors after PDT, and in particular, 12 h after PDT, the expression levels were much higher as compared with those in the LLC tumors. The results of our studies using in vitro and in vivo models suggest that the cell death caused by PDT was inhibited by induction of GADD-45alpha expression and that tumor recurrence was promoted by the enhancement of VEGF expression mediated by IL-2 upregulation. Therefore, it is speculated that the use of an IL-2 inhibitor may improve the efficacy of NPe6-PDT.     

Herpes simplex virus thymidine kinase gene transduction enhances tumor growth rate and cyclooxygenase-2 expression in murine colon cancer cells

Transduction of tumor cells with herpes simplex virus thymidine kinase (HSV-tk) gene and subsequent treatment with the prodrug ganciclovir (GCV) is the most common system utilized to date for "suicide" gene therapy of cancer. In the current report, we show that HSV-tk gene transduction enhances tumor growth rate of murine colon cancer cells, that are implanted subcutaneously in syngeneic mice, and enhances cyclooxygenase-2 (COX-2) protein expression and prostaglandin E(2) (PGE(2)) release in vitro and in vivo. It is further shown that the observed phenomenon is related to the presence of the HSV-tk sequence insert in the retroviral vector used for HSV-tk gene delivery. Transduction of murine colon cancer cells with control vector, carrying the neomycin-resistance gene alone, failed to increase tumor growth rate and COX-2 protein expression or PGE(2) production. On the contrary, it even decreased tumor growth, COX-2 protein expression and PGE(2.) The growth rate of HSV-tk-transduced murine tumors was significantly reduced by treatment with the selective COX-2 inhibitor nimesulide. Additionally, we demonstrate herein that both enhanced growth rate of HSV-tk-transduced murine tumors and increased levels of PGE(2) in HSV-tk-transduced cells persist upon the development of GCV resistance. Taken together, these results provide a better understanding of the direct effect of HSV-tk gene transduction on tumor cell biology and target tumor development.     

舒林酸、尼美舒利对人胃癌SGC-7901 细胞增殖及COX-2 、NF-κB 表达的抑制作用

 目的 探讨舒林酸、尼美舒利对人胃癌SGC-7901细胞增殖及COX-2、NF-κB表达的影响. 方法 对人胃癌SGC-7901细胞采用细胞培养,MTT法检测不同浓度舒林酸、尼美舒利对人胃癌SGC-7901细胞增殖抑制作用,免疫组化、western blot方法检测药物对COX-2、NF-κB表达的影响. 结果 舒林酸、尼美舒利可以抑制人胃癌SGC-7901细胞生长并具有浓度、时间依赖性;与阴性对照组相比,药物作用48h后尼美舒利100、200μmol/L组,舒林酸0.6、1.2mmol/L组COX-2、NF-κB蛋白表达明显减弱并具有剂量依赖性（P＜0.05）,COX-2、NF-κB之间正相关（P＜0.05）. 结论 舒林酸、尼美舒利可以抑制人胃癌SGC-7901细胞增殖,抑制NF-κB, COX-2的蛋白表达在二者抑制肿瘤机制中可能具有一定作用.     

选择性环氧合酶2抑制剂对人膀胱癌细胞T24增殖和凋亡的作用

背景与目的:环氧合酶2(cyclooxygenase-2,COX-2)与膀胱癌的发生发展密切相关,因此COX-2抑制剂对于膀胱癌可能具有潜在的抗肿瘤效应.本实验旨在探讨选择性COX-2抑制剂对人膀胱癌细胞T24增殖和凋亡的抑制作用.方法:用四甲基偶氮唑蓝还原法(MTT)研究选择性COX-2抑制剂SC-58125、塞来昔布(celecoxib)和非选择性COX抑制剂吲哚美辛(indomethacin)对T24细胞增殖的影响;用流式细胞术、DNA电泳和Hoechst33258荧光染色3种方法检测SC-58125作用下T24细胞的凋亡,同时逆转录聚合酶链反应(RT-PCR)检测细胞凋亡相关基因Bcl-2和Bax基因的变化.结果:在12.5 μmol/L至200 μmol/L的剂量范围内,SC-58125、塞来昔布及吲哚美辛均能不同程度地抑制T24细胞的增殖,以SC-58125的抑制作用最强,IC50在25 μmol/L～50 μmol/L之间;3种凋亡检测方法均观察到SC-58125作用后凋亡细胞的比例增加,其中100 μmol/L的SC-58125作用于T24细胞6 h和12 h后,流式细胞仪测得凋亡细胞的比例分别为(7.95±1.88)%和(12.5±2.42)%,较对照组增加(P＜0.05),但细胞内与凋亡相关的Bcl-2和Bax基因表达没有变化.结论:选择性COX-2抑制剂体外可抑制T24细胞的增殖,并诱导细胞凋亡.     

Enhancement of the effect of 5-aminolevulinic acid-based photodynamic therapy by simultaneous hyperthermia

In the present study, we have investigated a combination of photodynamic therapy (PDT) using 5-aminolevulinic acid (ALA) and simultaneous hyperthermia (HT) on osteosarcoma (HOSM-1) cells, squamous cell carcinoma (KB) cells and fibroblasts (HF), including an assessment of the differences in the sensitivity of these cells to such treatment in vitro. The intracellular accumulation of protoporphyrin IX (PPIX) formed by metabolism of ALA in mitochondria and the influence of the treatment on the mitochondrial membrane potential were evaluated using flow cytometry. The antitumor effects of HT, PDT using ALA (ALA-PDT) and ALA-PDT combined with HT (PDT+HT) were determined by an MTT assay. Western blot analysis of the expression of heat shock protein 60 (Hsp60) and Hsp70 was performed to evaluate the mitochondrial stress caused by each treatment. The intracellular PPIX accumulation in HOSM-1 cells was about 2-fold higher than that in KB cells. An antitumor effect of ALA-PDT and PDT+HT was obtained in each cell line, and indicated a synergistic interaction of the combination therapy in tumor cells. A marked degree of depolarization of the mitochondrial membrane was observed in both tumor cell lines, and there was no marked difference in the degree of depolarization between the cell lines. Marked expression of Hsp60 was observed in HOSM-1 cells treated with PDT+ HT and ALA-PDT, but not in KB cells. Slightly increased expression of Hsp70 was observed for all treatments in both tumor cell lines. These results suggest that the antitumor effect of ALA-PDT therapy against malignant tumor cells is enhanced by simultaneous HT. Furthermore, the differences in sensitivity to these therapies between the two cell types may have occurred because PPIX was not effectively utilized in HOSM-1 cells, compared to its utilization in KB cells.