Imatinib exhibit synergistic pleiotropy in the prevention of colorectal cancer by suppressing proinflammatory, cell survival and angiogenic signaling.

Recent global incidences and mortality rates have placed colorectal cancer (CRC) at third and second positions, respectively, among both sexes of all ages. Resistance during chemotherapy is a big problem in the treatment and disease-free survival of CRC patients. Discovery of new anticancer drug(s) is a time taking process and therefore, invites studies for repurposing the known therapeutics. The present study was conceived to analyze the anticancer role of Imatinib in experimental CRC at early stages. Different experimental procedures e.g. tumor incidences or histoarchitectural changes, gene and protein expression analysis, estimations of intracellular calcium, ROS, mitochondrial membrane potential, apoptotic index and molecular docking was performed to support the hypothesis. It was observed that Imatinib could function as an immunomodulator by breaking the feed-back loop between the proinflammatory cytokines (IL-1ß and TNF-α) and transcription factors (NF-κB, Jak3/Stat3) knowingly involved in increased cell proliferation during tumorigenesis via activating different intracellular signaling. Also, Imatinib could independently deregulate the other cell survival and proliferation signaling e.g. PI3-K/Akt/mTOR, Wnt/ß-catenin and MAPK. Proinflammatory cytokines orchestrated intracellular signaling also involve angiogenic factors to be upregulated during CRC which were also seemed to be independently suppressed by Imatinib. Restoration of physiological apoptosis by increasing the release of intracellular calcium to generate ROS thereby reducing the mitochondrial membrane potential for the release of cytochrome c and activation of caspase-3 was also reported with Imatinib administration. Thus, it may be suggested that Imatinib show synergistic pleiotropy in suppressing the interlinked tumorigenic signaling pathways independently.

Imatinib modulates pro-inflammatory microenvironment with angiostatic effects in experimental lung carcinogenesis.

Lung cancer has second highest rate of incidence and mortality around the world. Smoking cigarettes is the main stream cause of lung carcinogenesis along with other factors such as spontaneous mutations, inactivation of tumor suppressor genes. The present study was aimed to identify the mechanistic role of Imatinib in the chemoprevention of experimental lung carcinogenesis in rat model. Gross morphological observations for tumor formation, histological examinations, RT-PCR, Western blotting, fluorescence spectroscopy and molecular docking studies were performed to elucidate the chemopreventive effects of Imatinib and support our hypothesis by various experiments. It is evident that immuno-compromised microenvironment inside solid tumors is responsible for tumor progression and drug resistance. Therefore, it is inevitable to modulate the pro-inflammatory signaling inside solid tumors to restrict neoangiogenesis. In the present study, we observed that Imatinib could downregulate the inflammatory signaling and also attributed angiostatic effects. Moreover, Imatinib also altered the biophysical properties of BAL cells such as plasma membrane potential, fluidity and microviscosity to restrict their infiltration and thereby accumulation to mount immuno-compromised environment inside the solid tumors during angiogenesis. Our molecular docking studies suggest that immunomodulatory and angiostatic properties of Imatinib could be either independent of each other or just a case of synergistic pleiotropy. Imatinib was observed to activate the intrinsic or mitochondrial pathway of apoptosis to achieve desired effects in cancer cell killings. Interestingly, binding of Imatinib inside the catalytic domain of PARP-1 also suggests that it has caspase-independent properties in promoting cancer cell deaths.

The Role of IL-33/ST2 Pathway in Tumorigenesis.

Cancer is initiated by mutations in critical regulatory genes; however, its progression to malignancy is aided by non-neoplastic cells and molecules that create a permissive environment known as the tumor stroma or microenvironment (TME). Interleukin 33 (IL-33) is a dual function cytokine that also acts as a nuclear factor. IL-33 typically resides in the nucleus of the cells where it is expressed. However, upon tissue damage, necrosis, or injury, it is quickly released into extracellular space where it binds to its cognate receptor suppression of tumorigenicity 2 (ST2)L found on the membrane of target cells to potently activate a T Helper 2 (Th2) immune response, thus, it is classified as an alarmin. While its role in immunity and immune-related disorders has been extensively studied, its role in tumorigenesis is only beginning to be elucidated and has revealed opposing roles in tumor development. The IL-33/ST2 axis is emerging as a potent modulator of the TME. By recruiting a cohort of immune cells, it can remodel the TME to promote malignancy or impose tumor regression. Here, we review its multiple functions in various cancers to better understand its potential as a therapeutic target to block tumor progression or as adjuvant therapy to enhance the efficacy of anticancer immunotherapies.

Jagged-2 (JAG2) enhances tumorigenicity and chemoresistance of colorectal cancer cells.

Colorectal cancer (CRC) is one of the leading causes of cancer-related mortality. Recent studies have stated that NOTCH signaling plays an important role in the development and progression of CRC. However, the role of Jagged-2 (JAG2), one of the NOTCH ligands, has not been delineated in colorectal tumorigenesis and drug resistance. In the present study, we have examined the impact of targeting JAG2 on CRC cells. Among all the members of NOTCH ligands, only the expression of JAG2 was found up-regulated in the intestinal tumors of Apc Min /+ mice as compared to the nearby normal mucosa. JAG2 expression was also observed in a panel of human CRC cell lines. Pharmacological inhibition or genetic knockdown of ß-catenin in CRC cell lines suppressed JAG2 expression, suggesting Wnt/ß-catenin regulation of JAG2 expression. In addition, deletion of Apc gene in the intestinal cells of Apc conditional knockout mice resulted in up-regulation of JAG2 expression. Modulation of JAG2 expression significantly affected in vivo tumorigenicity of CRC cell lines. Moreover, knockdown of JAG2 sensitized CRC cells to chemotherapeutic agents, while ectopic expression of JAG2 increased chemoresistance of the CRC cells. Significant down-regulation of p21 was observed in JAG2-knockdown cells. Forced expression of p21 rescued the sensitivity of JAG2-knockdown cells to doxorubicin. In addition, the chemosensitivity of p21-null cells was not affected by JAG2 knockdown. These results suggest that JAG2 modulates the sensitivity of CRC cells to chemotherapeutic agents through p21. Our study identifies JAG2 as a novel target for therapeutic intervention of CRC.

Transgenic expression of cyclooxygenase-2 (COX2) causes premature aging phenotypes in mice.

Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostanoids, lipid signaling molecules that regulate various physiological processes. COX2, one of the isoforms of COX, is highly inducible in response to a wide variety of cellular and environmental stresses. Increased COX2 expression is thought to play a role in the pathogenesis of many age-related diseases. COX2 expression is also reported to be increased in the tissues of aged humans and mice, which suggests the involvement of COX2 in the aging process. However, it is not clear whether the increased COX2 expression is causal to or a result of aging. We have now addressed this question by creating an inducible COX2 transgenic mouse model. Here we show that post-natal expression of COX2 led to a panel of aging-related phenotypes. The expression of p16, p53, and phospho-H2AX was increased in the tissues of COX2 transgenic mice. Additionally, adult mouse lung fibroblasts from COX2 transgenic mice exhibited increased expression of the senescence-associated ß-galactosidase. Our study reveals that the increased COX2 expression has an impact on the aging process and suggests that modulation of COX2 and its downstream signaling may be an approach for intervention of age-related disorders.

Lentivirus-mediated somatic recombination and development of a novel mouse model for sporadic colorectal cancer.

In this study, we have developed a novel mouse model for sporadic colorectal cancer (CRC) by utilizing APC conditional knockout (Apc(CKO) ) mouse and lentivirus encoding Cre recombinase and a reporter gene (EGFP or LacZ). Lentiviral transduction of colonic crypt stem cells allowed for the long-term expression of reporter gene as well as excision of floxed Apc alleles, which resulted in tumor development. Tumors represented adenoma stages along with the nuclear accumulation of ß-catenin. Loss of E-cadherin at the cellular junctions and strong expression of Vimentin suggested the sign of active epithelial-mesenchymal transition. Moreover, nuclear staining of Ki67 inside epithelial cells of aberrant crypts demonstrated their higher proliferative nature. Erratic downstream signaling of activated Wnt/ß-catenin, AKT/mTOR, and Notch pathways provided strong evidence towards the higher proliferative index of epithelial cells inside the aberrant crypts. These results do recapitulate the findings of previous APC mutant mouse models. Our model represents sporadic CRC more precisely as (i) tumors result from somatic mutations but not from germline; (ii) tumors develop in colon not in small intestine; (iii) few tumors develop at the distal end of colons. Additionally, our model allows for the long-term expression of the gene(s), which get integrated into the host cell genome and provides an ability to track the tumor growth. © 2016 Wiley Periodicals, Inc.

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.

Epigenetic therapy for colorectal cancer.

Aberrations in epigenome that include alterations in DNA methylation, histone acetylation, and miRNA (microRNA) expression may govern the progression of colorectal cancer (CRC). These epigenetic changes affect every phase of tumor development from initiation to metastasis. Since epigenetic alterations can be reversed by DNA demethylating and histone acetylating agents, current status of the implication of epigenetic therapy in CRC is discussed in this article. Interestingly, DNA methyltransferase inhibitors (DNMTi) and histone deacetylase inhibitors (HDACi) have shown promising results in controlling cancer progression. The information provided here might be useful in developing personalized medicine approaches.

Sulindac and Celecoxib regulate cell cycle progression by p53/p21 up regulation to induce apoptosis during initial stages of experimental colorectal cancer.

In the present study we have elaborated the putative mechanisms could be followed by the non-steroidal anti-inflammatory drugs (NSAIDs) viz. Sulindac and Celecoxib in the regulation of cell cycle checkpoints along with tumor suppressor proteins to achieve their chemopreventive effects in the initial stages of experimental colorectal cancer. Male Sprague-Dawley rats were administered with 1,2-dimethylhydrazine dihydrochloride (DMH) to produce early stages of colorectal carcinogenesis. The mRNA expression profiles of various target genes were analyzed by RT-PCR and validated by quantitative real-time PCR, whereas protein expression was analyzed by Western blotting. Nuclear localization of transcription factors or other nuclear proteins was analyzed by electrophoretic mobility shift assay and immunofluorescence. Flowcytometry was performed to analyze the differential apoptotic events and cell cycle regulation. Molecular docking studies with different target proteins were also performed to deduce the various putative mechanisms of action followed by Sulindac and Celecoxib. We observed that DMH administration has abruptly increased the proliferation of colonic cells which is macroscopically visible in the form of multiple plaque lesions and co-relates with the disturbed molecular mechanisms of cell cycle regulation. However, co-administration of NSAIDs has shown regulatory effects on cell cycle checkpoints via induction of various tumor suppressor proteins. We may conclude that Sulindac and Celecoxib could possibly follow p53/p21 mediated regulation of cell proliferation, where down regulation of NF-κB signaling and activation of PPARγ might serve as important additional events in vivo.

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.

Role of cytokines and Jak3/Stat3 signaling in the 1,2-dimethylhydrazine dihydrochloride-induced rat model of colon carcinogenesis: early target in the anticancer strategy.

The molecular mechanisms by which colon cancer cells regulate the expression of various proinflammatory and anti-inflammatory cytokines and transcription factors resulting in tumor progression have not been well clarified. The present study thus explores the effect of cancer cell-derived cytokines and transcription factors on the chemoprevention of a rat model of early colon carcinogenesis. Elevated expression of proinflammatory cytokines [interleukin-1ß (IL-1ß), IL-2, interferon γ, and tumor necrosis factor-α] and the transcription factors [Janus kinase 3 (Jak3) and signal transducer and activator of transcription 3 (Stat3)] was found in the 1,2-dimethylhydrazine dihydrochloride (DMH) group; however, this elevated expression was reversed by the individual and combination treatment with piroxicam, a traditional nonsteroidal anti-inflammatory drug [inhibiting both cyclooxygenase-1 (COX-1) and COX-2] and c-phycocyanin, a cyanobacterium-derived biliprotein from Spirulina platensis (selective COX-2 inhibitor). In the DMH group, low expression of IL-4, an anti-inflammatory cytokine, was further observed with respect to the other groups. Expression of inducible nitric oxide synthase and nitric oxide/citrulline levels was also analyzed and was found to be elevated with DMH treatment. Increased apoptotic index and stimulated levels of Bcl-2-associated death promoter (Bad), a proapoptotic protein, were observed in piroxicam-treated and c-phycocyanin-treated rats. In-silico molecular docking of piroxicam as a ligand with several regulatory proteins was performed, indicating that, except inducible nitric oxide synthase, it effectively binds with COX-1, COX-2, Jak3, and Stat3. Piroxicam and c-phycocyanin perhaps showed chemopreventive properties by inhibiting proinflammatory cytokines and Jak3/Stat3 signaling while promoting apoptosis. In addition, a combination regimen was found to be more beneficial than monotherapy.

NSAIDs may regulate EGR-1-mediated induction of reactive oxygen species and non-steroidal anti-inflammatory drug-induced gene (NAG)-1 to initiate intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer.

This study aims to investigate the unclear molecular relationship involved in the activation of intrinsic pathway of apoptosis and NSAID-activated gene-1 (NAG-1) induction as a putative target in NSAIDs-mediated chemoprevention of colorectal cancer. Male Sprague-Dawley rats were administered with a colon-specific pro-carcinogen, 1,2-dimethylhydrazine dihydrochloride to achieve the early stages of colorectal cancer. Histopathological examination was performed for the analysis of neoplastic lesions while flow cytometry was performed for the relative quantification of intracellular reactive oxygen species (ROS), differential mitochondrial membrane potential (MMP or ΔΨ(M)), and apoptotic events. Various target biomolecules were analyzed either for their mRNA or protein expression profiles via RT-PCR and quantitative Real-Time PCR, or Western blotting and immunofluorescence, respectively. Enhanced gene as well as protein expression of pro-apoptotic agents was observed with the daily oral administration of two NSAIDs viz. Sulindac (cyclooxygenase (COX)-non-specific) and Celecoxib (a selective COX-2 inhibitor). A significant increase in early growth response-1 (EGR-1) protein expression and nuclear localization in NSAIDs co-administered animals may have positively regulated the expression of NAG-1 with a significant enhancement of intracellular ROS in turn decreasing the ΔΨ(M) to initiate apoptosis. In silico molecular docking analysis also showed that Sulindac and Celecoxib can block the active site pocket of B-cell lymphoma-extra large (Bcl-xL, anti-apoptotic transmembrane mitochondrial protein) which could be a putative mechanism followed by these NSAIDs to overcome anti-apoptotic properties of the molecule. NSAIDs-mediated up-regulation of EGR-1 and thereby NAG-1 along with implication of higher ROS load may positively regulate the intrinsic pathway of apoptosis for the chemoprevention of colorectal cancer.

Role of cytokines in experimentally induced lung cancer and chemoprevention by COX-2 selective inhibitor, etoricoxib.

This study explored the role of pro- and anti-inflammatory cytokines in dimethyl benz(a)anthracene (DMBA)-induced lung cancer and its subsequent correction with a COX-2 inhibitory NSAID, etoricoxib. A single dose of DMBA (20 mg/kg body weight) in 0.9 % NaCl administered intratracheally was used to induce tumors in the rat lungs in 20 weeks. The study of pro-inflammatory cytokines like IL-1ß, TNF-α, and IFN-γ revealed their upregulation by DMBA administration and restoration of their levels toward normal by the treatment with etoricoxib, while the anti-inflammatory cytokine IL-2 was found to be down-regulated with carcinogen administration and corrected with etoricoxib treatment. Apoptosis was studied by mitochondrial Bcl-2/Bax ratio and staining with fluorescent dyes acridine orange/ethidium bromide. The results showed a decreased apoptotic level with DMBA which was corrected with etoricoxib. Also, mitochondrial membrane potential was studied using JC-1 and rhodamine-123, which are membrane permeant fluorescent dyes, and generate information about cells at lower and higher mitochondrial membrane potential (∆Ψ(M)). The results showed the presence of maximum number of cells with higher ∆Ψ(M) in the DMBA group and their number was considerably lowered in the other three groups.

Up-regulation of p53 and mitochondrial signaling pathway in apoptosis by a combination of COX-2 inhibitor, Celecoxib and Dolastatin 15, a marine mollusk linear peptide in experimental colon carcinogenesis.

Programmed cell death, also known as apoptosis, is an active process occurring in eukaryotic cells and it depends on various sets of pro and anti-apoptotic proteins. Chemoprevention of colorectal cancer can be achieved by inducing apoptosis using synthetic compound, Celecoxib and natural peptide, Dolastatin 15 in an effective manner. But the apoptotic signaling by these two drugs remain unclear. The present study was thus focused on the role of Bcl2 family of proteins and their interplay with p53 in rats during the chemoprevention by these two drugs. After treatment for 6 wk with 1, 2-dimethylhydrazine (DMH), animals showed a marked occurrence of multiple plaque lesions. However, a simultaneous treatment with Celecoxib and Dolastatin 15 decreases such number to a significant level. DMH treatment also decreases the number of apoptotic cells in the colonic enterocytes which were corrected to the normal level by Celecoxib and Dolastatin 15. An increased expression of Bcl2 while other proteins like Bax, Apaf-1, cyt c, and caspases in the apoptotic pathway, and the tumor suppressor proteins, p53 and p21 get down-regulated after DMH treatment which were reverted back to normal with Celecoxib and Dolastatin 15. Also, cells having high mitochondrial membrane potential had been seen to increase to significant levels which were reduced after the administration of these anti-inflammatory drugs. In silico molecular docking studies also showed that Dolastatin 15 and Celecoxib may bind to the active site pocket of Bcl2 , thus revealing the direct target of Dolastatin 15 and Celecoxib apart from binding to COX-2.

Angiostatic properties of sulindac and celecoxib in the experimentally induced inflammatory colorectal cancer.

Initiation of various cancers has been observed to be regulated via a prolonged inflammatory state in the tissues. However, molecular role of such a localized inflammation is not clear in the advanced stages of colorectal cancer. In this study, we have elaborated the role of various pro- and anti-inflammatory cytokines, transcription, and angiogenic factors in the progression of the 1,2-dimethylhydrazine dihydrochloride (DMH)-induced late phage colorectal cancer and also observed the chemopreventive role of the two non-steroidal anti-inflammatory drugs (NSAIDs), viz., Sulindac and Celecoxib. Carcinogenic changes were observed with morphological and histopathological studies, whereas mRNA and protein regulations of various biomolecules were identified via RT- or qRT-PCR, western blot and immunofluorescence analysis, respectively. Activity of inducible nitric oxide (NO) and cyclooxygenase-2 enzymes were analyzed using standard NO assay and prostaglandin E2 immunoassay, whereas activities of matrix metalloproteinases (MMP-2 and-9) were identified by gelatin zymography. Flowcytometry was performed for the relative quantification of the apoptotic events. Molecular docking studies of Sulindac and Celecoxib were also performed with different target proteins to observe their putative mechanisms of action. As a result, we found that DMH-treated animals were having over-expression of various pro-inflammatory cytokines (IL-1ß, IL-2, and IFNγ), aberrant nuclear localization of activated cell survival transcription factors (NF-κB and Stat3) along with the increased incidence of activated angiogenic factors (MMP-2 and MMP-9) suggesting a marked role of inflammation in the tumor progression. However, NSAIDs co-administration has significantly reduced the angiogenic potential of the growing neoplasm.

Dolastatin 15, a mollusk linear peptide, and Celecoxib, a selective cyclooxygenase-2 inhibitor, prevent preneoplastic colonic lesions and induce apoptosis through inhibition of the regulatory transcription factor NF-κB and an inflammatory protein, iNOS.

The marine ecosystem is a unique and enormously rich source of natural products with potential chemopreventive applications in cancer. In the present study, we explored the chemopreventive role and the molecular mechanism of Dolastatin, a linear peptide from an Indian Ocean mollusk, and Celecoxib, a well-established cyclooxygenase-2 (COX-2) inhibitor in an individual as well as in a combination regimen in 1,2-dimethylhydrazine dihydrochloride (DMH)-induced colon carcinogenesis in a rat model. After a 6-week treatment with DMH, morphological analysis revealed a marked occurrence of preneoplastic features in the colonic mucosa, whereas histologically well-characterized dysplasia and hyperplasia were observed in DMH-treated animals. Simultaneous administration of Celecoxib and Dolastatin reduced these features significantly. DMH treatment affected the number of apoptotic cells in colonic enterocytes, which reverted to the normal level with the use of Celecoxib and Dolastatin. Inflammation remains the dominant molecular mechanism in the development of multiple plaque lesions, the carcinogenic lesions in a DMH-induced process that may be mediated by COX-2. Western blot and immunofluorescence analysis revealed a higher expression of COX-2 and nuclear factor-κB, the transcription factors responsible for proinflammatory proteins such as TNFα, and also the inducible nitric oxide synthase in the DMH group, which was further recovered significantly with the use of Celecoxib and Dolastatin. In-silico molecular docking analysis of Dolastatin as a ligand with various regulatory proteins suggests that although the peptide failed to dock to COX-2, it successfully did so with inducible nitric oxide synthase, thereby indicating the potential of this inflammatory protein as a molecular anticancer target in colon carcinogenesis.

Role of Sulindac and Celecoxib in the regulation of angiogenesis during the early neoplasm of colon: exploring PI3-K/PTEN/Akt pathway to the canonical Wnt/ß-catenin signaling.

Angiogenesis refers to the generation of new blood vasculature from the nearby pre-existing one and is regulated by a balance between the pro- and anti-angiogenic factors. During carcinogenesis, pro-angiogenic factors dominate and initialize the growth of new blood capillaries to provide nutrition, growth factors and overcome hypoxia inside the tumor microenvironment. In the present study, we have observed the role of Phosphatidylinositol-3-kinase (PI3-K)/Phophatase and tensin homolog deleted on chromosome ten (PTEN)/Akt (Protein kinase B) pathway and canonical Wnt/ß-catenin downstream signaling in the regulation of various pro-angiogenic molecules including the vascular endocrine growth factor-A (VEGF-A), matix metalloproteinases (MMPs), inducible nitric oxide synthase (iNOS) and chemokines for the progression of experimental colorectal cancer with 1,2-dimethylhydrazine dihydrochloride (DMH) and anti-angiogenic effects of two non-steroidal anti-inflammatory drugs (NSAIDs) viz. Sulindac and Celecoxib. Morphological and histopathological studies were performed to analyze the tumorigenic modifications while flow cytometry for the relative quantification of apoptotic events. Transcriptional and translational modifications of biomolecules were analyzed via Reverse Transcriptase-and quantitative Real Time PCR, Western immoblotting and immunoflurescence, respectively. In vitro phosphorylation, gelatin zymography and nitric oxide (NO) assay were performed to observe the activation states of Akt, MMPs and iNOS enzyme, respectively. Dysregultion in Akt activation, and thereby, aberrant signaling of ß-catenin along with the production of NO could positively regulate tumor angiogenesis. NSAIDs can overcome these carcinogenic effects by controlling various key check points including higher PTEN and glycogen synthase kinase-3ß (GSK-3ß) expression and repressing Akt, MMPs and iNOS activation while inducing apoptosis among the cancer cells.

Role of Sulindac and Celecoxib in chemoprevention of colorectal cancer via intrinsic pathway of apoptosis: exploring NHE-1, intracellular calcium homeostasis and Calpain 9.

Non-steroidal anti-inflammatory drugs (NSAIDs) have become promising agents for the chemoprevention of colorectal cancer presumably by inducing apoptosis. However, the role of NSAIDs in the regulation of NHE-1, intracellular calcium [Ca(2+)](i) homeostasis and Calpain 9 signaling pathway seems unclear. Male Sprague-Dawley rats were used as model for experimental colorectal cancer. Effects of pro-carcinogen (DMH) and NSAIDs were studied via morphological examination of tumors, histopathological changes, [Ca(2+)](i) homeostasis, production of reactive oxygen species (ROS), changes in mitochondrial membrane potential (MMP or ΔΨ(M)) and expression profile of target genes and proteins both at transcription and translation levels respectively. Size and number of tumors were found less in NSAIDs co-administered groups as compared to the DMH alone. Higher expression of NHE-1 was observed in DMH group whereas Calpain 9 was up-regulated in NSAIDs co-administered groups. [Ca(2+)](i) levels and reactive oxygen species were observed elevated in NSAIDs co-administered groups. ΔΨ(M) was found higher in DMH alone group along with the increased expression of the anti-apoptotic and mitochondrial membrane guard protein, Bcl-2. Expression levels of various pro-apoptotic proteins were observed higher in case of NSAIDs co-administered groups. Down-regulation of NHE-1, along with an increased [Ca(2+)](i) and induction of intrinsic pathway of apoptosis via activated Calpain 9 could be a putative mechanism pursued by Sulindac and Celecoxib for the chemoprevention of colorectal cancer.

Chemopreventive effects of NSAIDs as inhibitors of cyclooxygenase-2 and inducers of apoptosis in experimental lung carcinogenesis.

Roles of cyclooxygenase (COX) enzyme and intrinsic pathway of apoptosis have been explored for the chemopreventive effects of non-steroidal anti-inflammatory drugs (NSAIDs) on 9,10-dimethyl benz(a)anthracene (DMBA)-induced lung cancer in rat model. 16 weeks after the administration of DMBA, morphological analysis revealed the occurrences of tumours and lesions, which were regressed considerably with the co-administration of indomethacin and etoricoxib, the two NSAIDs under investigation. DMBA group was marked by hyperplasia and dysplasia as observed by histological examination, and these features were corrected to a large extent by the two NSAIDs. Elevated levels of COX-2 were seen in the DMBA group, the enzyme responsible for prostaglandin synthesis during inflammation and cancer, whilst the expression of the constitutive isoform, COX-1, was equally expressed in all the groups. Apoptosis was quantified by studying the activities of apaf-1, caspase-9, and 3 by immunofluorescence and western blots. Their activities were found to diminish in the DMBA-treated animals as compared to the other groups. Fluorescent co-staining of the isolated broncho-alveolar lavage cells showed reduced number of apoptotic cells in the DMBA group, indicating decrease in apoptosis after carcinogen administration. The present results thus suggest that the mechanism of cancer chemoprevention of NSAIDs may include the suppression of COX-2 and the induction of apoptosis.