Design, synthesis and antitumor activity of novel pyran-functionalized uracil derivatives: docking studies.

Aim: Synthesis of novel pyran-based uracils that may have potent antitumor activity against hepatocellular carcinoma HepG2 and ovarian cancer SKOV3 cell lines. Materials & methods: Novel pyran-based uracils were synthesized and their anticancer activity was assessed using methyl thiazolyl tetrazolium and wound-healing assays to detect their cytotoxicity and their antiproliferative and antimigratory activities. Results: Compounds 3, 5, 6, 7, 8, 9, 10, 11 and 13 significantly inhibited cell proliferation of the HepG2 cell line. Compounds 7, 8, 9 and 13 significantly inhibited the proliferation of SKOV3 cells, which was also proven through docking studies with topoisomerase I. Conclusion: The molecular docking analysis revealed that 7 and 9 are two major compounds found to possess higher degrees of interaction with DNA gyrase.

Constant light exposure and/or pinealectomy increases susceptibility to trichloroethylene-induced hepatotoxicity and liver cancer in male mice.

Exposure to light at night, pineal gland impairment, and the environmental pollutant trichloroethylene (TCE) have serious implications for health and contribute to illness, including liver cancer. The adverse effect of the association of continuous exposure to light with decreased melatonin levels and TCE-induced toxicity is not disclosed in target organs. This work explored the role of light and pineal impairment in increasing susceptibility to liver toxicity and cancer upon exposure to TCE. Male albino mice were divided into groups as follows: control group (12-h light/12-h dark cycle), constant light (24-h light), pinealectomized (Pnx) mice, sham surgically treated group, TCE-treated groups subjected to two doses (500 and 1000 mg/kg) at two different light regimens, and combination of Pnx and TCE-treated mice kept at a 12-h light/12-h dark cycle. Melatonin levels were significantly decreased in both Pnx mice and TCE-treated animals at both light regimens. Aspartate transaminase, alanine aminotransferase, activities, and serum bilirubin levels were significantly elevated, whereas albumin levels were markedly decreased in Pnx mice, TCE-treated mice, and the combination group. Histopathological investigations reflected changes in liver function parameters indicating liver injury and induction of cancer. These effects were accompanied by significant increase of the liver cancer biomarker alpha-fetoprotein and the expression of the metastatic markers CD44, TGFß-1, and VEGF, along with increased oxidative stress indicators and inflammatory cytokines (IL-6, IL-1ß, and TNF-α) in both Pnx and TCE-treated mice and the combination group at both light regimens. Taken together, our findings indicated that low melatonin levels, exposure to constant light, and the combination of both factors increases susceptibility to the toxic and carcinogenic effects of TCE on the liver.

Impact of the phytochemicals cocktail "breast safeguard" in regulating the interplay between redox signalling and murine adenocarcinoma cell proliferation, survival and angiogenesis.

Phytochemicals are natural plant extracts with a potent antioxidant, anti-inflammatory and anticancer characteristics by acting as a cell signalling modulator. This study aims to evaluate the effect of a commercial cocktail of phytochemicals "Breast safeguard" (BSG) in upregulating the expression of antioxidant enzymes to counteract signalling pathways that promote Ehrlich cells progression. The potent antioxidant activity and total phenolics and flavonoids contents of BSG was chemically validated, BSG treated mice showed a significant reduction at the tumor size, along with significant reduction in the expression of prognostic markers CEA and TNFα and induction of cell cycle arrest at G1/S phase as well as downregulation of Ki67. BSG supplementation significantly diminished H2O2, NO, MDA levels and upregulated the expression of SOD, CAT, GPx and GSH antioxidant enzymes in plasma and tumor tissues. BSG treatment markedly activated P53/Bax/Bcl2/c-caspase 3 signalling for cell apoptosis and attenuated the expression of antiapoptotic survivin protein. Meanwhile, BSG significantly diminished the expression of VEGF as an indication of angiogenesis inhibition. In conclusion, BSG exerted a significant upregulation of antioxidant enzymes which may be involved in upregulating P53/Bax/c-caspase 3 expression and attenuation of cell proliferation and angiogenesis.

Melatonin: A regulator of the interplay between FoxO1, miR96, and miR215 signaling to diminish the growth, survival, and metastasis of murine adenocarcinoma.

Melatonin (Mel.), also known as the magic hormone, is a nocturnally secreted hormone orchestrates the clearance of free radicals that have been built up and cumulated during day. This study aims to detect the impact of pineal gland removal on the incidence of tumor development and to assess the signaling pathways via which exogenous melatonin counteract cancer growth. This goal has been achieved by novel approach for pineal destruction using dental micromotor which validated by melatonin downregulation in blood plasma. Mice were injected sub-cutenously with Ehrlich cells to develop solid tumor as a murine model of breast cancer. The increase at tumor markers carcino embryonic antigen, TNFα, and nuclear factor kappa-light-chain-enhancer of activated B cells was over countered by exogenous melatonin supplementation (20 mg/kg) daily for 1 month. The anticancer effects of melatonin were significantly mediated by scavenging H2 O2 and NO and diminishing of lipid peroxidation marker malondialdehyde. The real-time polymerase chain Rx analyses indicated a significant effect of Melatonin in upregulating the expression of miR215, fork head box protein O1 (foxO1), and downregulation of miR96. Flowcytometric analyses indicated a significant effect of melatonin on induction of cell cycle arrest at G1 phase which was further confirmed by Ki67 downregulation. Immunohistochemical analyses indicated the role of melatonin in upregulating P53-dependent apoptosis and downregulating CD44 signaling for survivin, matrix metallo-protein kinase 2, and vascular endothelial growth factor to inhibit cell survival and metastasis. In conclusion, this study sheds the light on M./P53/miR215/CD44 with an emphasis on M./miR96//foxO1 signaling cascades, as a novel pathway of melatonin signaling in adenocarcinoma to diminish cancer cell growth, survival and metastasis.

Radio-sensitizing effect of a cocktail of phytochemicals on HepG2 cell proliferation, motility and survival.

Radio-resistance is a major hurdle challenging oncologist worldwide. Despite their anti-cancer characteristics, the implication of phytochemicals in clinical trials is still limited. This study is designed to evaluate the anticancer characteristics and radio-sensitizing effect of a cocktail of seven phytochemicals on HepG2 cells. Characterization of phytochemicals combination phenolic and flavonoids content as well as their scavenging activity were tested. The effective concentration of BSG that will be used as a radio-sensitizing dose was calculated using AlamarBlue assay. Treatment of HepG2 cells with BSG and/or ionizing radiations led to significant downregulation at cell proliferation as indicated by the decrease of colony formation ratio, proliferation marker (Ki67) expression as well as G2/M cell cycle arrest. The combined treatment stimulated P53-dependent apoptosis which was indicated by the significant increase of early apoptosis marker (Annexin V) expression, DNA fragmentation, expression of P53 & Bax and downregulation of Bcl2 expression. Combined treatment significantly attenuated HepG2 cell motility which was validated using wound healing migration assay and the significant reduction at CD95 expression. This study demonstrates the anti-cancer effect of BSG and its fundamental role in provoking cell responsiveness to IR leading to a significant inhibition at HepG2 cell proliferation, survival and migration.

The anti-tumourigenic effect of ellagic acid in SKOV-3 ovarian cancer cells entails activation of autophagy mediated by inhibiting Akt and activating AMPK.

This study investigated the effect of ellagic acid (EA) on SKOV-3 cell growth and invasiveness and tested if the underlying mechanism involves modulating autophagy. Cells were treated with EA in the presence or absence of chloroquine (CQ), an autophagy inhibitor, compound C (CC), an AMPK inhibitor, or an insulin-like growth factor-1 (IGF-1), a PI3K/Akt activator. EA, at an IC50 of 36.6 µmol/L, inhibited cell proliferation, migration, and invasion and induced cell apoptosis in SKOV-3 cells. These events were prevented by CQ. Also, EA increased levels of Beclin-1, ATG-5, LC3I/II, Bax, cleaved caspase-3/8 and reduced those of p62 and Bcl-2 in these cancer cells. Mechanistically, EA decreased levels of p-S6K1 (Thr389 ) and 4EBP-1 (Thr37/46 ), two downstream targets of mTORC1, and p-Akt (Thr308 ) but increased levels of AMPK (Thr172 ) and p-raptor (Ser792 ), a natural inhibitor of mTORC1. CC or IGF-1 alone partially prevented the effect of EA on cell survival, cell invasions, and levels of LDH, Beclin-1, and cleaved caspase-3. In conclusion, EA can inhibit SKOV-3 growth, migration, and invasion by activating cytotoxic autophagy mediated by inhibition of mTORC1 and Akt and activation of AMPK.

Chemoprevention of rat mammary carcinogenesis by spirulina.

Spirulina (SP) (Arthrospira platensis; previously Spirulina platensis) is a filamentous blue-green microalga (cyanobacterium) with potent dietary phytoantioxidant and anticancerous properties. We investigated the chemopreventive effect of SP against 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat breast carcinogenesis, and further studied its underlying mechanisms of action in vitro. Remarkably, SP cleared DMBA-induced rat mammary tumors, which was clearly confirmed by morphological and histological methods. SP supplementation reduced the incidence of breast tumors from 87% to 13%. At the molecular level, immunohistochemical analysis revealed that SP supplementation reduced expression of both Ki-67 and estrogen α. More interestingly, molecular analysis in the in vitro experiments indicated that SP treatment inhibited cell proliferation by 24 hours, which was accompanied by increased p53 expression, followed by increased expression of its downstream target gene, Cdkn1a (alias p21 or p21(Waf1/Cip1)). In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48 hours after SP treatment. To our knowledge, this is the first report of in vivo chemopreventive effect of SP against DMBA-induced breast carcinogenesis in rat, supporting its potential use in chemoprevention of cancer.

TGF-ß2: A Novel Target of CD44-Promoted Breast Cancer Invasion.

We have developed a tetracycline (tet)-off regulated expression of CD44s gene in the breast cancer (BC) cell line MCF-7 (B5 clone) and identified TGF-ß2 (Transforming Growth Factor beta-2; 3 fold induction) as a potential CD44-downstream transcriptional target by microarray analysis. To further validate this finding, the same RNA samples, used for microarray analysis and their corresponding protein lysates, collected from the BC cell line MCF-7-B5, were examined for CD44 expression in the presence of HA. Our results showed that TGF-ß2 mRNA levels were significantly elevated following the removal of tetracycline at 18, 24, and 48 h post-HA stimulation compared to the parental cells. Furthermore, the TGF-ß2 precursor protein increased in a time-dependent pattern upon HA-stimulation and in the absence of tetracycline. More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-ß2 expression upon HA-stimulation, and subsequently inhibited BC cell invasion in vitro. In addition to identifying TGF-ß2 as a target for HA/CD44 signaling, this data suggests that ATF/CREB might be a potential transcription factor linking HA/CD44 activation to TGF-ß2 transcription and additional experiments are required for a better understanding of the molecular mechanisms underpinning the novel function of the CD44/ TGF-ß2 signaling pathway in breast cancer metastasis.

Survivin is a novel target of CD44-promoted breast tumor invasion.

The hyaluronan (HA) receptor CD44 plays an essential role in cell-cell or cell-extracellular matrix communications and is a bioactive signal transmitter. Although a number of studies have described the function of CD44 in breast cancer (BC) metastasis, the underlying mechanisms have yet to be determined. By using a validated tetracycline-off-regulated CD44 expression system in the MCF-7 cell line combined with microarray analysis, we identified survivin (SVV) as a potential downstream transcriptional target of CD44. To test the hypothesis that SVV underpins CD44-promoted BC cell invasion, we combined molecular and pharmacologic approaches and showed that CD44 induction increased SVV expression levels, which in turn promotes BC cell invasion. Further, clinical analysis of breast tissue samples showed that SVV expression patterns paralleled those of the standard form of CD44 during breast tumor progression. More interestingly, we identified the PI3K/E2F1 pathway as a potential molecular link between HA/CD44 activation and SVV transcription. In addition to identifying SVV as a target for HA/CD44 signaling, this investigation provides a better understanding of the molecular mechanisms that underpin the novel function of SVV in breast cancer metastasis.

Chemoprevention of rat liver toxicity and carcinogenesis by Spirulina.

Spirulina platensis (SP) is a filamentous cyanobacterium microalgae with potent dietary phyto-antioxidant, anti-inflammatory and anti-cancerous properties. The present study aimed to investigate the chemopreventive effect of SP against rat liver toxicity and carcinogenesis induced by dibutyl nitrosamine (DBN) precursors, and further characterized its underlying mechanisms of action in HepG2 cell line. Investigation by light and electron microscopy showed that DBN treatment induced severe liver injury and histopathological abnormalities, which were prevented by SP supplementation. The incidence of liver tumors was significantly reduced from 80 to 20% by SP. Immunohistochemical results indicated that both PCNA and p53 were highly expressed in the liver of DBN-treated rats, but were significantly reduced by SP supplementation. Molecular analysis indicated that SP treatment inhibited cell proliferation, which was accompanied by increased p21 and decreased Rb expression levels at 48hrs post-treatment. In addition, SP increased Bax and decreased Bcl-2 expression, indicating induction of apoptosis by 48hrs. This is the first report of the in vivo chemopreventive effect of SP against DBN-induced rat liver cytotoxicity and carcinogenesis, suggesting its potential use in chemoprevention of cancer.

Towards understanding the mode of action of the multifaceted cell adhesion receptor CD146.

CD146, also known as melanoma cell adhesion molecule or MCAM, is a key cell adhesion protein in vascular endothelial cell activity and angiogenesis. CD146 promotes tumor progression of many cancers including melanoma and prostate. Strikingly, its expression is frequently lost in breast carcinoma cells, and it may act as a suppressor of breast cancer progression. While upstream mechanisms regulating CD146 are well documented, our understanding of the downstream molecular events underlying its mode of action remains to be elucidated. This review aims to focus on the progress in understanding the signaling mechanisms and the functional relevance of CD146, a multifaceted molecule, in cancer with particular emphasis on its role in inhibiting breast cancer progression.

Characterization of coordinated immediate responses by p16INK4A and p53 pathways in UVB-irradiated human skin cells.

While the precise mechanisms of melanoma development are unknown, recent in vivo studies have revealed that the p16(Ink4a)/Rb pathway is disrupted in melanomagenesis. Here, we characterize the role of p16/Rb in coordinating the early events in UVB-irradiated skin. Foreskins and melanoma cell cultures were irradiated with low and high acute UVB doses and examined for cell-cycle- and apoptosis-associated genes. In melanoma cells, low UVB dose upregulated p16, p53, and p21 expression levels in Malme-3M, and high UVB dose accentuated the expression of p53 and p21(Cip1/Waf1), in particular; however, in SkMel-28 cells only p16 expression was upregulated in response to UV irradiation. In HaCaT cells, high UVB dose caused dramatic increase in p53 expression followed by upregulation of p21(Cip1/Waf1) and Bax, and downregulation of Bcl-2 leading to apoptosis. In HaCaT cells, reinstatement of p16 pathway restored cell-cycle arrest in response to low dose. Foreskin organ culture experiments confirmed our in vitro cell results. These data indicate that the p53 and p16 pathways respond independently to UVB insult. The p16 pathway is favored at low doses and results in cell-cycle arrest; the p53 pathway is more responsive to higher doses and induces apoptosis depending on p53 mutation status.

In vivo evidence for the role of CD44s in promoting breast cancer metastasis to the liver.

The hyaluronan receptor CD44 plays an important role in facilitating invasion and metastasis of a variety of tumors, including breast carcinomas. CD44 functions as a bioactive signaling transmitter. Although a number of studies have implicated CD44 in breast tumor invasion, the evidence is still circumstantial. We have developed a tetracycline-regulated CD44s (standard form) system in the weakly metastatic breast cancer cell MCF7, which exhibits low endogenous expression of CD44 and generated a new cell line, MCF7F-B5. Induction of CD44s alone affected the growth characteristics of MCF7F-B5 cells by increasing their abilities to proliferate, migrate, and invade in vitro. In addition, we have identified and validated cortactin as a novel transcriptional target of hyaluronan/CD44s signaling in underpinning breast tumor invasion. To test these observations in vivo, we developed a doxycycline (DOX)-regulated CD44s breast cancer xenograft model. Induction of CD44s did not affect the growth rate or local invasion of the primary tumor. However, although no mice from the +DOX group developed metastasis, 8 of 11 mice from the -DOX group developed secondary tumors to the liver only. Interestingly, metastatic breast tumors expressed high levels of CD44. This study provides in vivo evidence for the role of the standard form of CD44 in promoting breast tumor invasion and metastasis to the liver.