Chemopreventive action of Imatinib, a tyrosine kinase inhibitor in the regulation of angiogenesis and apoptosis in rat model of lung cancer.

The present study explored the events of angiogenesis and apoptosis in 7,12-dimethyl benz(a)anthracene (DMBA)-induced lung cancer in rat and its chemoprevention with Imatinib, a receptor tyrosine kinase inhibitor. Further, it includes  lipopolysaccharide (LPS) mediating inflammation along with DMBA for the promotion of lung carcinogenesis. The animals received a single intratracheal instillation of DMBA (20 mg/kg body weight) in olive oil and LPS (0.6 mg/kg body weight) to induce tumors in 16 weeks. Besides morphology and histology of the lung tissues, RT-PCR, western blots, and immunofluorescence were performed for the expression of apoptotic and angiogenic proteins. Apoptosis was studied by mitochondrial Bcl-2/Bax ratio and staining with the dyes Acridine orange/ethidium bromide of the isolated Broncho epithelial cells. Also, mitochondrial membrane potential (ΔΨM) was studied by JC-1. The study revealed that the expression of VEGF, MMP-2, MMP-9, and the chemokine MCP-1 to be very high in DMBA and DMBA + LPS groups, while Bcl-2 also shows an elevated expression. These results were restored with Imatinib treatment. The pro-apoptotic proteins, Bax, Bad, Apaf-1, and Caspase-3 were highly diminished in DMBA and DMBA + LPS groups which were recovered with Imatinib treatment.

Role of GSK-3ß in Regulation of Canonical Wnt/ß-catenin Signaling and PI3-K/Akt Oncogenic Pathway in Colon Cancer.

Non-steroidal anti-inflammatory drugs (NSAIDs) are emerging as novel chemopreventive agents because of their ability in blocking cellular proliferation, and thereby tumor development, and also by promoting apoptosis. GSK-3ß, a serine threonine kinase and a negative regulator of the oncogenic Wnt/ß-catenin signaling pathway, plays a critical role in the regulation of oncogenesis. Celecoxib and etoricoxib, the two cyclooxygenase-2 (COX-2) selective NSAIDs, and Diclofenac, a preferential COX-2 inhibitory NSAID, had shown uniformly the chemopreventive and anti-neoplastic effects in the early stage of colon cancer by promoting apoptosis as well as an over-expression of GSK-3ß while down-regulating the PI3-K/Akt oncogenic pathway.

Sesamol Induces Apoptosis by Altering Expression of Bcl-2 and Bax Proteins and Modifies Skin Tumor Development in Balb/c Mice.

BACKGROUND: Chemoprevention using natural agents has emerged as a new and promising strategy for reducing cancer burden. Sesamol, a water soluble lignin, is a potent antioxidant with potential anticancer activities. Its small size (molecular weight: 138.34g) coupled with easy permeability (log P: 1.29) results in its excessive systemic loss therefore, compromising local bioavailability. Furthermore, irritant nature of sesamol limits its application on skin per se. OBJECTIVE: Present study aims to evaluate chemopreventive efficacy of free and encapsulated (SLNs) sesamol, at gross and molecular level, in DMBA induced skin cancer animal model. METHODS: Evaluation is done in terms of tumor burden quantification, histological evaluation of skin, determination of oxidative stress, and quantification of apoptotic proteins, bcl-2 and bax, using both western blot analysis and immunofluorescence studies. RESULTS: Sesamol administration (both in free and encapsulated form) significantly decreased the tumor burden and lipid peroxidation level and increased anti-oxidant levels, thereby hampering the development and promotion of skin tumors. Further, downregulation of bcl-2 and stimulation of bax protein expression on treatment with both free and encapsulated sesamol was responsible for the induction of apoptosis in tumor cells. Encapsulating sesamol into SLNs not only reduced its irritant nature which limits its direct topical application but also improved its local targeting to skin. CONCLUSION: Both free and encapsulated sesamol demonstrated the inhibition of tumor progression by inducing skin cell apoptosis via bcl-2/bax mediated pathway.

Chemopreventive action of non-steroidal anti-inflammatory drugs on the inflammatory pathways in colon cancer.

Non-steroidal anti-inflammatory drugs (NSAIDs) are emerging as novel chemopreventive agents against a variety of cancers owing to their capability in blocking the tumor development by cellular proliferation and by promoting apoptosis. Inflammation is principal cause of colon carcinogenesis. A missing link between inflammation and cancer could be the activation of NF-κB, which is a hallmark of inflammatory response, and is commonly detected in malignant tumors. Therefore, targeting pro-inflammatory cyclooxygenase enzymes and transcription factors will be profitable as a mechanism to inhibit tumor growth. In the present study, we have studied the role of various pro-inflammatory enzymes and transcription factors in the development of the 1,2-dimethylhydrazine dihydrochloride (DMH)-induced colorectal cancer and also observed the role of three NSAIDs, viz., Celecoxib, Etoricoxib and Diclofenac. Carcinogenic changes were observed in morphological and histopathological studies, whereas protein regulations of various biomolecules were identified by immunofluorescence analysis. Apoptotic studies was done by TUNEL assay and Hoechst/PI co-staining of the isolated colonocytes. It was found that DMH-treated animals were having an over-expression of pro-inflammatory enzymes, aberrant nuclear localization of activated cell survival transcription factor, NF-κB and suppression of anti-inflammatory transcription factor PPAR-γ, thereby suggesting a marked role of inflammation in the tumor progression. However, co-administration of NSAIDs has significantly reduced the inflammatory potential of the growing neoplasm.

Downregulation of telomerase activity by diclofenac and curcumin is associated with cell cycle arrest and induction of apoptosis in colon cancer.

Uncontrolled cell proliferation is the hallmark of cancer, and cancer cells have typically acquired damage to genes that directly regulate their cell cycles. The synthesis of DNA onto the end of chromosome during the replicative phase of cell cycle by telomerase may be necessary for unlimited proliferation of cells. Telomerase, a ribonucleoprotein enzyme is considered as a universal therapeutic target of cancer because of its preferential expression in cancer cells and its presence in 90 % of tumors. We studied the regulation of telomerase and telomerase reverse transcriptase catalytic subunit (TERT) by diclofenac and curcumin, alone and also in combination, in 1, 2-dimethylhydrazine dihydrochloride-induced colorectal cancer in rats. The relationship of telomerase activity with tumors suppressor proteins (p51, Rb, p21), cell cycle machinery, and apoptosis was also studied. Telomerase is highly expressed in DMH group and its high activity is associated with increased TERT expression. However, telomerase is absent or is present at lower levels in normal tissue. CDK4, CDK2, cyclin D1, and cyclin E are highly expressed in DMH as assessed by RT-PCR, qRT-PCR, Western blot, and immunofluorescence analysis. Diclofenac and curcumin overcome these carcinogenic effects by downregulating telomerase activity, diminishing the expression of TERT, CDK4, CDK2, cyclin D1, and cyclin E. The anticarcinogenic effects shown after the inhibition of telomerase activity by diclofenac and curcumin may be associated with upregulation of tumor suppressor proteins p51, Rb, and p21, whose activation induces the cells cycle arrest and apoptosis.

Downregulation of PI3-K/Akt/PTEN pathway and activation of mitochondrial intrinsic apoptosis by Diclofenac and Curcumin in colon cancer.

Phosphatidylinositol 3-kinase (PI3-K)/PTEN/Akt signaling is over activated in various tumors including colon cancer. Activation of this pathway regulates multiple biological processes such as apoptosis, metabolism, cell proliferation, and cell growth that underlie the biology of a cancer cell. In the present study, the chemopreventive effects have been observed of Diclofenac, a preferential COX-2 inhibitory non-steroidal anti-inflammatory drugs, and Curcumin, a natural anti-inflammatory agent, in the early stage of colorectal carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride in rats. The tumor-promoting role of PI3-K/Akt/PTEN signal transduction pathway and its association with anti-apoptotic family of proteins are also observed. Both Diclofenac and Curcumin downregulated the PI3-K and Akt expression while promoting the apoptotic mechanism. Diclofenac and Curcumin administration significantly increased the expression of pro-apoptotic Bcl-2 family members (Bad and Bax) while decreasing the anti-apoptotic Bcl-2 protein. An up-regulation of cysteine protease family apoptosis executioner, such as caspase-3 and -9, is seen. Diclofenac and Curcumin inhibited the Bcl-2 protein by directly interacting at the active site by multiple hydrogen bonding, as also evident by negative glide score of Bcl-2. These drugs stimulated apoptosis by increasing reactive oxygen species (ROS) generation and simultaneously decreasing the mitochondrial membrane potential (ΔΨ M). Diclofenac and Curcumin showed anti-neoplastic effects by downregulating PI3-K/Akt/PTEN pathway, inducing apoptosis, increasing ROS generation, and decreasing ΔΨ M. The anti-neoplastic and apoptotic effects were found enhanced when both Diclofenac and Curcumin were administered together, rather than individually.

Dolastatin, along with Celecoxib, stimulates apoptosis by a mechanism involving oxidative stress, membrane potential change and PI3-K/AKT pathway down regulation.

BACKGROUND: Phosphoinositide 3-kinase (PI3-K) is an important regulator of oncogenesis and apoptosis in various types of cancers including colon cancer. A combinatorial strategy of using Cyclooxygenase-2 inhibitor, Celecoxib and Dolastatin, a linear peptide from marine mollusks of Indian Ocean origin has shown anti-neoplastic effects in colon cancer in a rat model. METHODS: The signal transduction pathway of PI3-K/AKT and the downstream signaling proteins had been studied in an early stage of colon carcinogenesis (DMH induced) by gene and protein expression, apoptotic studies by colonocyte apoptotic bleb assay, intracellular calcium level by fluorescence spectrometry, mitochondrial membrane potential by Rhodamine 123 flow cytometry and Reactive oxygen species measurement. Molecular docking analysis was employed to study the interaction of oncogenic proteins and the ligand, Celecoxib and Dolastatin. RESULTS: Apoptotic cell index was lowered with DMH while both the drugs increased it and inhibited PI3-K and AKT expression. Docking studies revealed both the proteins targeted by the drugs via an ATP binding site. An increased expression of GSK-3ß, pro-apoptotic protein Bad, transcription factor Egr-1, tumor suppressor protein PTEN while a downregulation of G1-associated cell cycle protein, Cyclin D1 and increased intracellular calcium as well as reactive oxygen species were observed. Also, the number of cells having a higher mitochondrial membrane potential was lowered. CONCLUSION: Celecoxib and Dolastatin inhibited the tumor development through regulation of the PI3-K/AKT pathway which can act as a novel target for these drugs. GENERAL SIGNIFICANCE: The anti-cancer properties of Dolastatin, a peptide isolated from marine mollusks in colorectal cancer is shown.

Angiostatic role of the selective cyclooxygenase-2 inhibitor etoricoxib (MK0663) in experimental lung cancer.

Lung cancer was induced in Sprague-Dawley rats by a single intra-tracheal instillation of 9,10-dimethybenz(a)anthracene (DMBA) and evaluated the anti-angiogenic action of etoricoxib, which is a selective cyclooxygenase-2 (COX-2) inhibitor. The animals were divided into four groups. Group 1 (Control) received 0.9% (w/v) normal saline intra-tracheal and 0.5% (w/v) carboxymethyl cellulose per oral daily as the vehicle of the drug, Group 2 received DMBA (20 mg/kg) intra-tracheal once, Group 3 received a daily oral dose of etoricoxib (0.6 mg/kg bw) in addition to the DMBA while Group 4 received etoricoxib alone. Morphological and histological analysis confirmed the presence of lung tumors 20 weeks after the administration of DMBA. Expressions of COX-2, MMP-2, MMP-9, MCP-1, MIP-1ß and VEGF were studied by immunofluorescence, Western immunoblot and mRNA studies, which showed a higher expression of these proteins in the DMBA-treated animals but much lower in DMBA+etoricoxib. Gelatin zymography as applied for the detection of the extracellular protein degrading enzymes, matrix metalloproteinases showed more intense activity in DMBA-treated rats as compared to the other groups. Also, the isolated alveolar macrophages were stained with Merocyanine540 (MC540) to study the membrane fluidity and lipid packing effect. DMBA treatment resulted in a significant increase in the number of lung cells exhibiting a high intensity of MC540 staining, which was reduced by the co-administration of etoricoxib. Thus the effects of etoricoxib on the expression of the angiogenic proteins have been observed, which clearly shows an anti-angiogenic mechanism of action of etoricoxib in lung cancer chemoprevention.

Diclofenac, a selective COX-2 inhibitor, inhibits DMH-induced colon tumorigenesis through suppression of MCP-1, MIP-1α and VEGF.

Angiogenesis is a physiological process involving growth of new blood vessels from pre-existing ones; however, it also plays a critical role in tumor progression. It favors the transition from hyperplasia to neoplasia, that is, from a state of cellular multiplication to uncontrolled proliferation. Therefore targeting angiogenesis will be profitable as a mechanism to inhibit tumor's lifeline. Further, it is important to understand the cross-communication between vascular endothelial growth factor (VEGF)-master switch in angiogenesis and other molecules in the neoplastic and pro-inflammatory milieu. We studied the role of two important chemokines [monocyte chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-lα] alongwith VEGF and matrix metalloproteinases (MMPs) in non-steroidal anti-inflammatory drugs (NSAIDs)-induced chemopreventive effect in experimental colon cancer in rat. 1,2-Dimethylhydrazine (DMH, 30 mg/kg body weight, subcutaneously (s.c.) once-a-week) for 18 wk was used as pro-carcinogen and diclofenac (8 mg/kg body weight, orally daily) as the preferential cyclooxygenase-2 (COX-2) inhibitor. Expression of COX-2 and VEGF was found to be significantly elevated in the DMH-treated group as compared to the control, which was lowered notably by Diclofenac co-administration with DMH. Gelatin zymography showed prominent MMP-9 activity in the DMH-treated rats, while the activity was nearly absent in all the other groups. Expression of MCP-1 was found to be markedly increased whereas MIP-1α expression was found to be decreased in colonic mucosa from DMH-treated rats, which was reversed in the DMH + Diclofenac group. Our results indicate potential role of chemokines alongwith VEGF in angiogenesis in DMH-induced cancer and its chemoprevention with diclofenac.

Intrinsic mitochondrial membrane potential change and associated events mediate apoptosis in chemopreventive effect of diclofenac in colon cancer.

The present study explored the role of intrinsic mitochondrial membrane potential (delta psi M) in NSAID-induced apoptosis in the early stages of colon cancer. 1,2-Dimethylhydrazine dihydrochloride (DMH) was used to induce colon cancer and its chemoprevention was studied by diclofenac in a rat model. After 6 weeks of treatment with DMH (early stage), morphological analysis revealed a marked occurrence of preneoplastic features [i.e., mucosal plaque lesions (MPLs) in the colonic tissue]. Coadministration of diclofenac with DMH resulted in a significant reduction of these lesions, thereby proving the chemopreventive efficacy of diclofenac at the chosen anti-inflammatory dose. DMH treatment also led to a significant increase in delta psi M in the isolated colonocytes as assessed by JC-1 fluorescent staining, measured both by fluorescence microscopy and spectrofluorometerically. Further, there was seen a reduction in the number of cells showing low delta psi M, and hence monomer intensity of JC-1 by DMH treatment. To study the mechanism of these alterations in delta psi M in the present work, we studied the protein expression of important proapoptotic proteins, cytochrome c and Bax, by Western blot analysis and immunohistochemistry. DMH treatment reduced the mitochondrial translocation of Bax whereas cytochrome c was found to be located prominently in the mitochondria. Protein expression of antiapoptotic Bcl-2 was also studied in the colonic mucosa, which was expectedly found to be overexpressed after DMH treatment. Diclofenac treatment ameliorated the elevated delta psi M and its associated events to exert its chemopreventive action against early stages of colon cancer.

Effect of etoricoxib, a cyclooxygenase-2 selective inhibitor on aberrant crypt formation and apoptosis in 1,2 dimethyl hydrazine induced colon carcinogenesis in rat model.

Etoricoxib, a second generation selective cyclooxygenase-2 (COX-2) inhibitor had been studied for the chemopreventive response at its therapeutic anti-inflammatory dose in 1,2-dimethylhydrazine (DMH) induced colon carcinogenesis in rat model. Eight to ten weeks old male rats of Sprague-Dawley strain were divided into four groups. While group 1 served as control and received the vehicle of the drugs, group 2 and 3 were administered freshly prepared DMH in 1mM EDTA-saline (pH 7.0) (30 mg/kg body wt/week, subcutaneously). Group 3 was also given a daily treatment of etoricoxib (0.6 mg/kg body wt orally) while the group 4 received the same amount of etoricoxib only, prepared in 0.5% carboxymethyl cellulose. Animals were sacrificed at the end of 6 weeks, body weight recorded and the colons were subjected to macroscopic and histopathological studies. The maximum number of raised mucosal lesions called the multiple plaque lesions (MPL) were found in the DMH group which significantly reverted back in the DMH + etoricoxib group, while very few MPLs were recorded in the control and etoricoxib only group. Similarly, the number of aberrant crypt foci (ACF), the point of future carcinogenic growth, was recorded more in the DMH group and significantly less in the DMH + etoricoxib group. The histopathological analysis showed the presence of severe hyperplasia, occasional dysplasia and aggregates of lymphoid cells in the localized regions. Etoricoxib group showed near normal histological features with the crypt architecture and the surrounding stromal tissue remaining intact. To ascertain the molecular mechanism of such anti-carcinogenic features the colonocytes were isolated and studied in primary culture for the evidence of apoptosis by fluorescent staining and genotoxic changes by single cell gel electrophoresis assay (comet assay) which shows that the DMH treated animals produced much less apoptotic nuclei but more comet producing cell, while these features were reverted back with the etoricoxib treatment. The cytoplasmic expression of COX-2 protein was studied in paraffin sections of the colon by immunohistochemistry with COX-2 specific antibody which showed a very high presence of this inducible enzyme with the DMH group while in all other groups of animals it was not visible or weekly expressed. The anti-inflammatory effect of the drug, etoricoxib was also validated by a carrageenan-induced inflammation in rat model which showed an extremely high anti-inflammatory response within the dose range used in the present study. Also the growth profile of all the animals remained the same throughout the six week period of the investigation as there was no change in the body weight. It appears that apoptosis remains the dominant anti-proliferative end effect of this drug, mediated by an inhibition of the proinflammatory COX-2 isoform although further molecular probings are needed to arrive at a conclusive agreement in favor of the chemoprotective use of such drugs in colon cancers.

Induction of pulmonary carcinogenesis in Wistar rats by a single dose of 9, 10 dimethylbenz(a)anthracene (DMBA) and the chemopreventive role of diclofenac.

OBJECTIVES: To evaluate the chemopreventive efficacy of Diclofenac, a preferential cyclooxygenase-2 (COX-2) inhibiting non steroidal anti-inflammatory drug (NSAID) in the 9, 10 Dimethylbenz(a)anthracene (DMBA) induced experimental lung carcinogenesis. METHODS: Animals were divided into 4 groups. Control group received normal saline intratratracheally. DMBA group was given DMBA (20 mg/kg of body weight) in the similar manner. DMBA+Diclofenac group was given daily oral dose of Diclofenac (8 mg/kg of body weight) in addition to DMBA while the last group received Diclofenac only. Animals were sacrificed after 24 weeks. COX-2 expression was studied by immunohistochemistry (IHC) and Western immunoblotting. For apoptosis study DNA fragmentation on agarose gel and florescent staining of alveolar macrophages were done. RESULTS: The incidence and burden of tumor were reduced by the Diclofenac treatment. Diclofenac caused the reduction in the COX-2 levels which were increased in the DMBA treated group. It also caused the induction of apoptosis as seen by both techniques. CONCLUSION: From all these results it can be concluded that Diclofenac might have a chemopreventive role for lung carcinogenesis which is mediated by suppression of COX-2 enzyme and induction of apoptosis.

Alterations in membrane fluidity and dynamics in experimental colon cancer and its chemoprevention by diclofenac.

We examined the role of membrane fluidity and dynamics as important early events in the carcinogenic transformation of colonic epithelial cells. 1,2-Dimethylhydrazine dihydrochloride (DMH) was used to induce initial stages of colon cancer and diclofenac was used for chemoprevention. To determine alterations of membrane fluidity of rat colonic epithelial cells, fluidity (inverse of fluorescence polarization) and order parameter were studied with 1,6-diphenylhexatriene (DPH) polarization. Order parameter as well as fluorescence polarization was found to be significantly decreased, thus demonstrating an increase in the fluidity of the membrane. To further confirm the fluidity changes, microviscosity of the cell membrane was studied using pyrene excimer formation, which showed a significant decrease in microviscosity and hence elevated membrane fluidity (translational diffusion). The colonocytes were stained with merocyanine 540 (MC540) to further elaborate the changes in membrane fluidity and lipid packing. The increased number of colonocytes showing high MC540 fluorescence pointed towards the wide spaces and hence, high fluidity in the membrane after DMH treatment. Membrane dynamics studies, i.e., lipid phase separation and membrane phase state were carried out using N-NBD-PE and Laurdan, respectively. We saw a transition from the gel to a more liquid crystalline state of the membrane in the Laurdan experiment. Further more percentage quenching (%Q) value of N-NBD-PE showed less phase separation (or domain formation). Diclofenac co-administration with DMH was successful in reverting the changes observed, confirming the role of these anti-inflammatory drugs in considerable lipid affinity and consequently in the chemoprevention of early stages of colon cancer.

Chemopreventive effect of nonsteroidal anti-inflammatory drugs on 9,10-dimethylbenz[a]anthracene-induced lung carcinogenesis in mice.

Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, celecoxib, and etoricoxib were studied as chemopreventive agents in lung cancer in mice induced by 9,10-dimethylbenz[a]anthracene (DMBA). The animals were subjected to a single intratracheal instillation of DMBA by surgical intervention, while they were treated with oral NSAIDs daily at their following anti-inflammatory dose: aspirin 25 mg/kg, celicoxib 6 mg/kg, and etoricoxib 0.6 mg/kg body weight, respectively. The animals were sacrificed after 18 weeks of treatment. Results showed a significant incidence of pulmonary tumors, dysplastic changes in histopathology, and signs of inflammatory occurrence in the DMBA-treated animals, which were grossly reversed by the NSAIDs. A greater number of macrophages, neutrophils, and lymphocytes were seen in the bronchoalveolar lavage (BAL) smear while the inflammatory cell counts decreased in DMBA + NSAIDs groups. A significant increase in the drug-metabolizing enzymes viz. cytochrome p450, cytochrome b5, and glutathione-S-transferase was noted in the DMBA group, which was reverted back in the NSAID-treated mice. Similarly, the subcelluler enzymes were elevated in DMBA, but significantly fell in the NSAID groups. DMBA also caused a higher level of lipid peroxidation as well as the different antioxidant enzyme activity, which were corrected by the NSAIDs. A marked elevation was noticed in the total lipid composition and its individual constituents in the DMBA group, which was reverted back appreciably by the NSAIDs. The results suggest that the DMBA-induced lung tumor development in balb/c mice could be a reliable model to test the chemopreventive potential of the NSAIDs.

Chemoprevention of 1,2-dimethylhydrazine-induced colon carcinogenesis by a non-steroidal anti-inflammatory drug, etoricoxib, in rats: inhibition of nuclear factor kappaB.

Etoricoxib, a highly selective cyclooxygenase- 2 (COX-2) inhibitor (a non steroidal anti-inflammatory drug) used for the treatment of rheumatoid arthritis and osteoarthritis, has been newly marketed and studied for the chemopreventive response in the 1,2-dimethylhydrazine dihydrochloride (DMH) induced rat colon cancer model. Male Sprague-Dawley rats were divided into four groups. Group I served as the Control and received the vehicle treatment, while Groups 2 and 3 were administered freshly prepared DMH (30 mg/kg body weight, subcutaneously) in 1mM EDTA-saline (pH 7.0). Groups 3 and 4 received Etoricoxib (0.64 mg/kg body weight, orally) daily prepared in 0.5% carboxymethyl cellulose. After a 6 week treatment period, animals were sacrificed and the colons were subjected to macroscopic and histopathological studies. Well characterized pre-neoplastic features such as multiple plaque lesions (MPLs), aberrant crypts (ACs) and aberrant crypt foci (ACF) were found in the DMH group. The number was reduced in DMH + Etoricoxib group, while very few MPLs and ACFs were recorded in the Etoricoxib only group. Also, histologically well characterized dysplasia and hyperplasia were observed in DMH treated group. The simultaneous administration of DMH and Etoricoxib reduced these features. To study apoptosis, colonocytes were isolated by metal chelation from colonic sacs and studied by fluorescent staining. The DMH treated animals produced much less apoptotic nuclei as compared to the Control. The number of apoptotic nuclei was also found higher in the DMH + Etoricoxib group as well as in Etoricoxib only group. Studies of a nuclear transcription factor (NF-kB) and COX-2 by Western blot analysis and immunohistochemistry demonstrated expression of both to be elevated in the DMH treated group but reduced in the DMH + Etoricoxib group. Expression was also low in the Etoricoxib only group. It may be concluded that the drug, Etoricoxib, has the potential to reduce DMH induced colon cancer development.

Pulmonary carcinogenesis in mice with a single intratracheal instillation of 9, 10-dimethyl benz[a]anthracene.

A single intratracheal instillation of 9,10-dimethyl benz(a)anthracene (DMBA) at 3 different doses of 5, 10, and 20 mg/kg body weight to Balb/c mice for 12 weeks had caused a significant incidence of pulmonary tumors along with inflammatory changes. The number of macrophages in the broncho-alveolar lavage (BAL) fluid increased significantly, while the neutrophil and lymphocyte count as well as the protein content in the BAL fluid remained unchanged. A marked elevation in the lipid peroxidation product as well as the antioxidative enzymes were noted in the DMBA-treated group. The BAL fluid, which contains the surfactant membrane, was tested for rotational diffusion of the small hydrocarbon fluorophore, diphenyl hexatriene, and resulted in an enhanced fluorescence polarization and anisotropy value as well as the order parameter. DMBA treatment also altered the toxicity parameters, such as the lipid peroxidation, catalase, total protein, reduced glutathione, and alanine and amino transferase activities in the liver and kidney tissues. The results suggest that DMBA-induced lung tumor development in Balb/c mice could be an important model for the study of pathophysiology of BAL-fluid-associated surfactant and offers to test a variety of promising chemopreventive/chemotherapeutic agents.

Chemopreventive effects of nonsteroidal anti-inflammatory drugs on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats.

ABSTRACT The anticancer efficacy of two different classes of NSAIDs, the nonspecific cyclooxygenase (COX) inhibitor aspirin and the specific COX-2 inhibitor celecoxib, was examined at their therapeutic anti-inflammatory doses during 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis in a rat model. Eight to 10-week-old male rats of Sprague strain were divided into four groups. While group 1 served as control and received the vehicle of the drugs, groups 2, 3, and 4 were administered freshly prepared DMH in 1 mM EDTA saline (pH 7.0) (30 mg/kg body weight/week, subcutaneously). Groups 3 and 4 were also given a daily treatment of aspirin (60 mg/kg body weight, orally) and celecoxib (6 mg/kg body weight, orally), respectively, both prepared in carboxy-methyl cellulose. Animals were sacrificed at the end of 12 weeks and colons from different groups were subjected to macroscopic and histopathological studies, enzymatic activities of superoxide dismutase (SOD) and catalase (CAT), and determination of lipid peroxide level. The maximum number of raised mucosal lesions in proximal, middle, and distal regions of the colon was found in the DMH group alone, and the lowest number was found in the celecoxib-treated DMH group. Histological studies also showed the highest occurrence of dysplastic aberrant crypt foci (ACF) associated with enlarged lymphoid follicles in all the three portions of colon (i.e., proximal, middle, and distal). The aspirin-administered DMH group had lesser ACF in the proximal and middle portions and no ACF in the distal region. The celecoxib-administered DMH group showed no ACF in the middle region of the rat colon. DMH treatment induced lipid peroxidation and inhibited the activities of SOD and CAT. Both the aspirin- and celecoxib-treated DMH groups showed a marked lowering of the lipid peroxide level along with a significant enhancement of CAT activity when compared with the DMH-treated group. The results show that celecoxib was found to be more effective in reducing the ACF occurrence and aggregates of lymphoid tissue than the nonselective COX inhibitor aspirin, and suggests a possible chemoprevention modality in colon cancer. This may have important implications as COX-2 selective drugs at anti-inflammatory doses are better tolerated clinically than standard NSAIDs, thus making them potentially better chemopreventive agents in colon cancer.

Effect of monensin on the enzymes of oxidative stress, thiamine pyrophosphatase and DNA integrity in rat testicular cells in vitro.

Monensin, a sodium specific ionophore was evaluated for its in vitro effects on rat testis by studying changes at biochemical parameters as well as at the DNA level. It was observed that monensin produced marked alterations in the activities of various enzymes associated with the testicular functions. The significant inhibition of different enzymes of oxidative defense system points toward the generation of reactive oxygen species (ROS) by monensin treatment. The significant depletion of reduced glutathione and elevation in the level of lipid peroxidation further support the above findings. The significant inhibition of the activities of lactate dehydrogenase and adenosine triphosphatase shows the interference of monensin with the normal energy supply in spermatogenesis. Moreover, the significant increase in the activities of acid phosphatase and thiamine pyrophosphatase demonstrates the interference of monensin with the Golgi-lysosomal complex of the rat testis. Induced DNA fragmentation indicates towards the impact of monensin on the DNA integrity and apoptosis. Further studies are needed to understand the important molecular mechanisms responsible for these effects.

Effect of antioxidant vitamin E as a protective factor in experimental atherosclerosis in rhesus monkeys.

Atherosclerosis has been known for many years, yet its etiology remains unknown. Hypercholesterolemia is a major risk factor for atherosclerosis. The mechanism by which it triggers endothelial injury is not known. Since the role of the antioxidant vitamin E on experimental atherosclerosis is inconsistent, the present study was undertaken to evaluate platelet lipid peroxidation and the role of vitamin E (alpha-tocopherol) as protective factor in atherosclerosis in rhesus monkeys. A significant decrease in serum cholesterol and serum triglyceride levels was found in the group of animals which were reverted to stock diet along with vitamin E injections after 9 months of atherogenic diet feeding. Decreases in malonyldialdehyde levels and antioxidant enzyme activities were less significant in animals continued on an atherogenic diet feeding along with vitamin E as compared with animals fed a stock diet with vitamin E supplementation. The overall observations in this study suggest that antioxidant status and lipid peroxidation could be partly restored with vitamin E supplementation in experimental atherosclerosis. Damage to endothelial cells destroys their antithrombotic status and leads to fatal thrombosis. alpha-Tocopherol offers the best hope, but the question is how much of it should be administered for the prevention of atherosclerosis.

The role of oxidized HDL in monocyte/macrophage functions in the pathogenesis of atherosclerosis in Rhesus monkeys.

The effect of oxidative modification of high-density lipoprotein (HDL) was assessed by incubation of normal HDL (obtained from Rhesus monkeys fed a stock diet) with 5 microM CuSO4 at 37 degrees C for 12 h/24 h. The physicochemical properties of oxidized-HDL (Ox-HDL) were found to be affected in terms of lipid peroxidation, as observed by the increased level of thiobarbituric acid reactive substances (nmol MDA/mg HDL protein). The biological properties of HDL were altered, since a decrease in the efflux of free cholesterol into the medium was found in the presence of Ox-HDL24h compared with normal HDL (N-HDL). The binding, uptake and degradation of 125I-LDL by macrophages increased in the presence of Ox-HDL24h. The activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione-peroxidase) was reduced in monocytes in the presence of Ox-HDL. However, in the presence of N-HDL, the levels of antioxidant enzymes were maintained at a higher level than in the control (in the absence of HDL) monocytes. Furthermore, the number of monocytes adhered to aortic endothelium were found to be increased in the presence of Ox-HDL. These findings suggest that HDL is susceptible to oxidative modification. Since the parameters selected in the present study are involved in the pathogenesis of atherosclerosis, it can be postulated that the in vivo protection of HDL in atherosclerosis can be reversed in the circumstances in which HDL undergoes oxidative modification like low-density lipoprotein (LDL).