Inhibition of Cyp1a Protects Mice against Anthracycline Cardiomyopathy.

Background: Anthracyclines such as doxorubicin (Dox) are highly effective anti-tumor agents, but their use is limited by dose-dependent cardiomyopathy and heart failure. Our laboratory previously reported that induction of cytochrome P450 family 1 (Cyp1) enzymes contributes to acute Dox cardiotoxicity in zebrafish and in mice, and that potent Cyp1 inhibitors prevent cardiotoxicity. However, the role of Cyp1 enzymes in chronic Dox cardiomyopathy, as well as the mechanisms underlying cardioprotection associated with Cyp1 inhibition, have not been fully elucidated. Methods: The Cyp1 pathway was evaluated using a small molecule Cyp1 inhibitor in wild-type (WT) mice, or Cyp1-null mice ( Cyp1a1/1a2 -/- , Cyp1b1 -/- , and Cyp1a1/1a2/1b1 -/- ). Low-dose Dox was administered by serial intraperitoneal or intravenous injections, respectively. Expression of Cyp1 isoforms was measured by RT-qPCR, and myocardial tissue was isolated from the left ventricle for RNA sequencing. Cardiac function was evaluated by transthoracic echocardiography. Results: In WT mice, Dox treatment was associated with a decrease in Cyp1a2 and increase in Cyp1b1 expression in the heart and in the liver. Co-treatment of WT mice with Dox and the novel Cyp1 inhibitor YW-130 protected against cardiac dysfunction compared to Dox treatment alone. Cyp1a1/1a2 -/- and Cyp1a1/1a2/1b1 -/- mice were protected from Dox cardiomyopathy compared to WT mice. Male, but not female, Cyp1b1 -/- mice had increased cardiac dysfunction following Dox treatment compared to WT mice. RNA sequencing of myocardial tissue showed upregulation of Fundc1 and downregulation of Ccl21c in Cyp1a1/1a2 -/- mice treated with Dox, implicating changes in mitophagy and chemokine-mediated inflammation as possible mechanisms of Cyp1a-mediated cardioprotection. Conclusions: Taken together, this study highlights the potential therapeutic value of Cyp1a inhibition in mitigating anthracycline cardiomyopathy.

SENTRI: Single-Particle Energy Transducer for Radionuclide Injections for Personalized Targeted Radionuclide Cancer Therapy.

PURPOSE: Targeted radionuclide therapy (TRT), whereby a tumor-targeted molecule is linked to a therapeutic beta- or alpha-emitting radioactive nuclide, is a promising treatment modality for patients with metastatic cancer, delivering radiation systemically. However, patients still progress due to suboptimal dosing, driven by the large patient-to-patient variability. Therefore, the ability to continuously monitor the real-time dose deposition in tumors and organs at risk provides an additional dimension of information during clinical trials that can enable insights into better strategies to personalize TRT. METHODS AND MATERIALS: Here, we present a single beta-particle sensitive dosimeter consisting of a 0.27-mm3 monolithic silicon chiplet directly implanted into the tumor. To maximize the sensitivity and have enough detection area, minimum-size diodes (1 µm2) are arrayed in 64 × 64. Signal amplifiers, buffers, and on-chip memories are all integrated in the chip. For verification, PC3-PIP (prostate-specific membrane antigen [PSMA]+) and PC3-flu (PSMA-) cell lines are injected into the left and right flanks of the mice, respectively. The devices are inserted into each tumor and measure activities at 5 different time points (0-2 hours, 7-9 hours, 12-14 hours, 24-26 hours, and 48-50 hours) after 177Lu-PSMA-617 injections. Single-photon emission computed tomography/computed tomography scans are used to verify measured data. RESULTS: With a wide detection range from 0.013 to 8.95 MBq/mL, the system is capable of detecting high tumor uptake as well as low doses delivered to organs at risk in real time. The measurement data are highly proportional (R2 > 0.99) to the 177Lu-PSMA-617 activity. The in vivo measurement data agree well with the single-photon emission computed tomography/computed tomography results within acceptable errors (±1.5%ID/mL). CONCLUSIONS: Given the recent advances in clinical use of TRT in prostate cancer, the proposed system is verified in a prostate cancer mouse model using 177Lu-PSMA-617.

Low cost, high temporal resolution optical fiber-based γ-photon sensor for real-time pre-clinical evaluation of cancer-targeting radiopharmaceuticals.

Cancer radiopharmaceutical therapies (RPTs) have demonstrated great promise in the treatment of neuroendocrine and prostate cancer, giving hope to late-stage metastatic cancer patients with currently very few treatment options. These therapies have sparked a large amount of interest in pre-clinical research due to their ability to target metastatic disease, with many research efforts focused towards developing and evaluating targeted RPTs for different cancer types in in vivo models. Here we describe a method for monitoring real-time in vivo binding kinetics for the pre-clinical evaluation of cancer RPTs. Recognizing the significant heterogeneity in biodistribution of RPTs among even genetically identical animal models, this approach offers long-term monitoring of the same in vivo organism without euthanasia in contrast to ex vivo tissue dosimetry, while providing high temporal resolution with a low-cost, easily assembled platform, that is not present in small-animal SPECT/CTs. The method utilizes the developed optical fiber-based γ-photon biosensor, characterized to have a wide linear dynamic range with Lutetium-177 (177Lu) activity (0.5-500 µCi/mL), a common radioisotope used in cancer RPT. The probe's ability to track in vivo uptake relative to SPECT/CT and ex vivo dosimetry techniques was verified by administering 177Lu-PSMA-617 to mouse models bearing human prostate cancer tumors (PC3-PIP, PC3-flu). With this method for monitoring RPT uptake, it is possible to evaluate changes in tissue uptake at temporal resolutions <1 min to determine RPT biodistribution in pre-clinical models and better understand dose relationships with tumor ablation, toxicity, and recurrence when attempting to move therapies towards clinical trial validation.

Circulating hemopexin modulates anthracycline cardiac toxicity in patients and in mice.

Anthracyclines such as doxorubicin (Dox) are effective chemotherapies, but their use is limited by cardiac toxicity. We hypothesized that plasma proteomics in women with breast cancer could identify new mechanisms of anthracycline cardiac toxicity. We measured changes in 1317 proteins in anthracycline-treated patients (n = 30) and replicated key findings in a second cohort (n = 31). An increase in the heme-binding protein hemopexin (Hpx) 3 months after anthracycline initiation was associated with cardiac toxicity by echocardiography. To assess the functional role of Hpx, we administered Hpx to wild-type (WT) mice treated with Dox and observed improved cardiac function. Conversely, Hpx-/- mice demonstrated increased Dox cardiac toxicity compared to WT mice. Initial mechanistic studies indicate that Hpx is likely transported to the heart by circulating monocytes/macrophages and that Hpx may mitigate Dox-induced ferroptosis to confer cardioprotection. Together, these observations suggest that Hpx induction represents a compensatory response during Dox treatment.

Evaluating the effectiveness of anti-Nogo treatment in spinal cord injuries.

As humans, we cannot regenerate axons within the central nervous system (CNS), therefore, making any damage to it permanent. This leads to the loss of sensory and motor function below the site of injury and can be crippling to a person's health. Spontaneous recovery can occur from plastic changes, but it is minimal. The absence of regeneration is due to the inhibitory environment of the CNS as well as the inherent inability of CNS axons to form growth cones. Amongst many factors, one of the major inhibitory signals of the CNS environment is the myelin-associated Nogo pathway. Nogo-A, Nogo-B and Nogo-C (Nogo), stimulate the Nogo receptor, inhibiting neurite outgrowth by causing growth cones to collapse through activation of Rho Kinase (ROCK). Antibodies can be used to target this signalling pathway by binding to Nogo and thus promote the outgrowth of neuronal axons in the CNS. This use of anti-Nogo antibodies has been shown to upregulate CNS regeneration as well as drastically improve sensory and motor function in both rats and primates when coupled with adequate training. Here, we evaluate whether the experimental success of anti-Nogo at improving CNS regeneration can be carried over into the clinical setting to treat spinal cord injuries (SCI) and their symptoms successfully. Furthermore, we also discuss potential methods to improve the current treatment and any developmental obstacles.

Changes in Citric Acid Cycle and Nucleoside Metabolism Are Associated with Anthracycline Cardiotoxicity in Patients with Breast Cancer.

Anthracyclines and HER2-targeted antibodies are very effective for the treatment of breast cancer, but their use is limited by cardiotoxicity. In this nested case-control study, we assessed the role of intermediary metabolism in 38 women with breast cancer treated with anthracyclines and trastuzumab. Using targeted mass spectrometry to measure 71 metabolites in the plasma, we identified changes in citric acid and aconitic acid that differentiated patients who developed cardiotoxicity from those who did not. In patients with cardiotoxicity, the magnitude of change in citric acid at three months correlated with the change in left ventricular ejection fraction (LVEF) and absolute LVEF at nine months. Patients with cardiotoxicity also demonstrated more pronounced changes in purine and pyrimidine metabolism. Early metabolic changes may therefore provide insight into the mechanisms associated with the development of chemotherapy-associated cardiotoxicity.

What are the links between hypoxia and Alzheimer's disease?

Alzheimer's disease (AD) is the most common neurodegenerative disease. Histological characterization of amyloid plaques and neurofibrillary tangles in the brains of AD patients, alongside genetic studies in individuals suffering the familial form of the disease, has fueled the accumulation of the amyloid-ß protein as the initial pathological trigger of disease. Association studies have recently showed that cerebral hypoxia, via both genetic and epigenetic mechanisms, increase amyloid-ß deposition by altering expression levels of enzymes involved in the production/degradation of the protein. Furthermore, hypoxia has also been linked to neuronal and glial-cell calcium dysregulation through formation of calcium-permeable pores, dysregulated glutamate signaling, and intracellular calcium-store dysfunction. Hypoxia has also been strongly linked to neuroinflammation; however, this relationship to AD has not been thoroughly discussed in the literature. Here, we highlight and organize critical research evidence showing that in both hypoxic and AD brains, there are similarities in terms of 1) the substances mediating/modulating the neuroinflammatory environment and 2) the immune cells that drive the formation of these substances.

Proproliferative function of adaptor protein GRB10 in prostate carcinoma.

Growth factor receptor-binding protein 10 (GRB10) is a well-known adaptor protein and a recently identified substrate of the mammalian target of rapamycin (mTOR). Depletion of GRB10 increases insulin sensitivity and overexpression suppresses PI3K/Akt signaling. Because the major reason for the limited efficacy of PI3K/Akt-targeted therapies in prostate cancer (PCa) is loss of mTOR-regulated feedback suppression, it is therefore important to assess the functional importance and regulation of GRB10 under these conditions. On the basis of these background observations, we explored the status and functional impact of GRB10 in PCa and found maximum expression in phosphatase and tensin homolog (PTEN)-deficient PCa. In human PCa samples, GRB10 inversely correlated with PTEN and positively correlated with pAKT levels. Knockdown of GRB10 in nontumorigenic PTEN null mouse embryonic fibroblasts and tumorigenic PCa cell lines reduced Akt phosphorylation and selectively activated a panel of receptor tyrosine kinases. Similarly, overexpression of GRB10 in PTEN wild-type PCa cell lines accelerated tumorigenesis and induced Akt phosphorylation. In PTEN wild-type PCa, GRB10 overexpression promoted mediated PTEN interaction and degradation. PI3K (but not mTOR) inhibitors reduced GRB10 expression, suggesting primarily PI3K-driven regulation of GRB10. In summary, our results suggest that GRB10 acts as a major downstream effector of PI3K and has tumor-promoting effects in prostate cancer.-Khan, M. I., Al Johani, A., Hamid, A., Ateeq, B., Manzar, N., Adhami, V. M., Lall, R. K., Rath, S., Sechi, M., Siddiqui, I. A., Choudhry, H., Zamzami, M. A., Havighurst, T. C., Huang, W., Ntambi, J. M., Mukhtar, H. Proproliferatve function of adaptor protein GRB10 in prostate carcinoma.

Fisetin targets YB-1/RSK axis independent of its effect on ERK signaling: insights from in vitro and in vivo melanoma models.

The anti-proliferative activity of dietary flavonoid fisetin has been validated in various cancer models. Establishing its precise mechanism of action has proved somewhat challenging given the multiplicity of its targets. We demonstrated that YB-1 promotes epithelial-to-mesenchymal transition and its inhibition suppressed tumor cell proliferation and invasion. The p90 ribosomal S6 kinase (RSK), an important ERK effector, activates YB-1 to drive melanoma growth. We found that fisetin treatment of monolayer/3-D melanoma cultures resulted in YB-1 dephosphorylation and reduced transcript levels. In parallel, fisetin suppressed mesenchymal markers and matrix-metalloproteinases in melanoma cells. Data from cell-free/cell-based systems indicated that fisetin inhibited RSK activity through binding to the kinase. Affinity studies for RSK isoforms evaluated stronger interaction for RSK2 than RSK1. Competition assays performed to monitor binding responses revealed that YB-1 and RSK2 do not compete, rather binding of fisetin to RSK2 promotes its binding to YB-1. Fisetin suppressed YB-1/RSK signaling independent of its effect on ERK, and reduced MDR1 levels. Comparable efficacy of fisetin and vemurafenib for inhibiting melanoma growth was noted albeit through divergent modulation of ERK. Our studies provide insight into additional modes of regulation through which fisetin interferes with melanoma growth underscoring its potential therapeutic efficacy in disease progression.

Targeting epithelial to mesenchymal transition in prostate cancer by a novel compound, plectranthoic acid, isolated from Ficus microcarpa.

Epithelial-to-mesenchymal transition (EMT) plays a crucial role in prostate cancer (PCa) metastasis. This has led to a surge in the efforts for identification of safer and more effective compounds which can modulate EMT and consequently inhibiting migration and invasion of PCa cells. We reported previously that Plectranthoic acid (PA), a natural compound isolated from the extracts of Ficus microcarpa, has the capability to induce cell cycle arrest and apoptosis in PCa cells. Here, we determined the effects of PA on EMT, migration, and invasion of PCa cells. Inhibition of EMT induced by different mitogens was effectively inhibited by PA treatment with subsequent decrease in migration of PCa cells. Employing a PCa cell culture model of TGF-ß-induced EMT, we showed that PA has the ability to reverse EMT. PA treatment was associated with induction of epithelial markers and decrease in the expression of mesenchymal markers in PCa cells. Proteomic analysis identified Rac1 as the major cadherin signaling protein modulated with PA treatment. In silico studies indicated that PA docked to the CH domain of NEDD9 protein with an estimated free binding energy of -7.34 Kcal/moL. Our studies revealed significant inhibition of Rac1/NEDD9 pathway in PA treated cells thereby providing a molecular basis of the inhibitory effect of PA on PCa cell migration and invasion. In conclusion, our data suggest that PA should be investigated further as an adjuvant treatment in human PCa cells, given its potential as an anti-invasive agent.

Dietary flavonoid fisetin increases abundance of high-molecular-mass hyaluronan conferring resistance to prostate oncogenesis.

We and others have shown previously that fisetin, a plant flavonoid, has therapeutic potential against many cancer types. Here, we examined the probable mechanism of its action in prostate cancer (PCa) using a global metabolomics approach. HPLC-ESI-MS analysis of tumor xenografts from fisetin-treated animals identified several metabolic targets with hyaluronan (HA) as the most affected. Efficacy of fisetin on HA was then evaluated in vitro and also in vivo in the transgenic TRAMP mouse model of PCa. Size exclusion chromatography-multiangle laser light scattering (SEC-MALS) was performed to analyze the molar mass (Mw) distribution of HA. Fisetin treatment downregulated intracellular and secreted HA levels both in vitro and in vivo Fisetin inhibited HA synthesis and degradation enzymes, which led to cessation of HA synthesis and also repressed the degradation of the available high-molecular-mass (HMM)-HA. SEC-MALS analysis of intact HA fragment size revealed that cells and animals have more abundance of HMM-HA and less of low-molecular-mass (LMM)-HA upon fisetin treatment. Elevated HA levels have been shown to be associated with disease progression in certain cancer types. Biological responses triggered by HA mainly depend on the HA polymer length where HMM-HA represses mitogenic signaling and has anti-inflammatory properties whereas LMM-HA promotes proliferation and inflammation. Similarly, Mw analysis of secreted HA fragment size revealed less HMM-HA is secreted that allowed more HMM-HA to be retained within the cells and tissues. Our findings establish that fisetin is an effective, non-toxic, potent HA synthesis inhibitor, which increases abundance of antiangiogenic HMM-HA and could be used for the management of PCa.

Dietary flavonoid fisetin for cancer prevention and treatment.

Cancer remains a major public health concern and a significant cause of death worldwide. Identification of bioactive molecules that have the potential to inhibit carcinogenesis continues to garner interest among the scientific community. In particular, flavonoids from dietary sources are the most sought after because of their safety, cost-effectiveness, and feasibility of oral administration. Emerging data have provided newer insights into understanding the molecular mechanisms that are essential to identify novel mechanism-based strategies for cancer prevention and treatment. Dietary flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) found in many fruits and vegetables has been shown in preclinical studies to inhibit cancer growth through alteration of cell cycle, inducing apoptosis, angiogenesis, invasion, and metastasis without causing any toxicity to normal cells. Although data from in-vitro and in-vivo studies look convincing, well-designed clinical trials in humans are needed to conclusively determine the efficacy across various cancers. This review highlights the chemopreventive and therapeutic effects, molecular targets, and mechanisms that contribute to the observed anticancer activity of fisetin against various cancers.

Potent anti-proliferative, pro-apoptotic activity of the Maytenus royleanus extract against prostate cancer cells: evidence in in-vitro and in-vivo models.

Prostate cancer is a leading of cause of cancer related death in men. Despite intensive investment in improving early diagnosis, it often escapes timely detection. Mortality remains high in advanced stage prostate cancer where palliative care remains the only option. Effective strategies are therefore needed to prevent the occurrence and progression of the disease. Plant-derived compounds have been an important source of several clinically useful anti-cancer agents and offer an attractive approach against prostate cancer. We previously showed that the methanol extract of Maytenus royleanus (MEM) leaves and its fractions possess significant antioxidant activity with therapeutic potential against free-radical associated damages. The present study evaluated the anti-proliferative activity of MEM in the prostate cancer model system. Analysis of MEM and its various fractions revealed the presence of triterpenoids, flavonoids and tannins, conjugated to one or more polar groups and carbohydrate moieties. Further studies against known standards established the existence of caffeic acid and quercetin 3-rhamnoside in varying concentration in different MEM fractions. Time course analysis of MEM treated prostate cancer cells indicated significant decrease in cell viability, assessed by MTT and clonogenic survival assays. This was accompanied by G2 phase arrest of cell cycle, downregulation of cyclin/cdk network and increase in cdk inhibitors. MEM treated cells exhibited cleavage of Caspase-3 and PARP, and modulation of apoptotic proteins, establishing apoptosis as the primary mechanism of cell death. Notably MEM suppressed AR/PSA signaling both in prostate cancer cell cultures and in the in vivo model. Intraperitoneal injection of MEM (1.25 and 2.5 mg/ animal) to athymic nude mice implanted with androgen sensitive CWR22Rν1 cells showed significant inhibition in tumor growth and decreased serum PSA levels reciprocating in vitro findings. Taken together, our data suggest that MEM may be explored further for its potential therapeutic effects against prostate cancer progression in humans.

Dietary polyphenols in prevention and treatment of prostate cancer.

Prostate cancer is the most prevalent disease affecting males in many Western countries, with an estimated 29,480 deaths in 2014 in the US alone. Incidence rates for prostate cancer deaths have been decreasing since the early 1990s in men of all races/ethnicities, though they remain about 60% higher in African Americans than in any other group. The relationship between dietary polyphenols and the prevention of prostate cancer has been examined previously. Although results are sometimes inconsistent and variable, there is a general agreement that polyphenols hold great promise for the future management of prostate cancer. Various dietary components, including polyphenols, have been shown to possess anti-cancer properties. Generally considered as non-toxic, dietary polyphenols act as key modulators of signaling pathways and are therefore considered ideal chemopreventive agents. Besides possessing various anti-tumor properties, dietary polyphenols also contribute to epigenetic changes associated with the fate of cancer cells and have emerged as potential drugs for therapeutic intervention. Polyphenols have also been shown to affect post-translational modifications and microRNA expressions. This article provides a systematic review of the health benefits of selected dietary polyphenols in prostate cancer, especially focusing on the subclasses of polyphenols, which have a great effect on disease prevention and treatment.

MicroRNAs and photocarcinogenesis.

As a new class of sequence-specific regulators of gene expression, the microRNAs (miRNA) form a regulatory network with growth factors and transcription factors participating in various biological processes. It is now being recognized that the various key processes involved in cancer induction are under the control of these small noncoding RNAs, which regulate ~30% of all human genes by targeting sequences in their 3'-untranslated regions. Photocarcinogenesis is a complex interplay of signaling events in the UV exposed human skin including DNA damage and repair, apoptosis, cell survival, mutations and the immune system. In this review, we have scrutinized the role of miRNAs in skin cancer biology focusing on the three most common types of skin cancer namely the basal cell carcinoma, squamous cell carcinoma and cutaneous malignant melanoma. An overview of these studies will be useful in gaining insights into the mechanisms of cancer development in the human skin. A better understanding of the functionality of miRNAs will have enormous implications to risk assessment, and to target interventions against signaling events involved in photocarcinogenesis.

Role of epithelial mesenchymal transition in prostate tumorigenesis.

Globally, the cancer associated deaths are generally attributed to the spread of cancerous cells or their features to the nearby or distant secondary organs by a process known as metastasis. Among other factors, the metastatic dissemination of cancer cells is attributed to the reactivation of an evolutionary conserved developmental program known as epithelial to mesenchymal transition (EMT). During EMT, fully differentiated epithelial cells undergo a series of dramatic changes in their morphology, along with loss of cell to cell contact and matrix remodeling into less differentiated and invasive mesenchymal cells. Many studies provide evidence for the existence of EMT like states in prostate cancer (PCa) and suggest its possible involvement in PCa progression and metastasis. At the same time, the lack of conclusive evidence regarding the presence of full EMT in human PCa samples has somewhat dampened the interest in the field. However, ongoing EMT research provides new perspectives and unveils the enormous potential of this field in tailoring new therapeutic regimens for PCa management. This review summarizes the role of many transcription factors and other molecules that drive EMT during prostate tumorigenesis.

Involvement of ER stress and activation of apoptotic pathways in fisetin induced cytotoxicity in human melanoma.

The prognosis of malignant melanoma remains poor in spite of recent advances in therapeutic strategies for the deadly disease. Fisetin, a dietary flavonoid is currently being investigated for its growth inhibitory properties in various cancer models. We previously showed that fisetin inhibited melanoma growth in vitro and in vivo. Here, we evaluated the molecular basis of fisetin induced cytotoxicity in metastatic human melanoma cells. Fisetin treatment induced endoplasmic reticulum (ER) stress in highly aggressive A375 and 451Lu human melanoma cells, as revealed by up-regulation of ER stress markers including IRE1α, XBP1s, ATF4 and GRP78. Time course analysis indicated that the ER stress was associated with activation of the extrinsic and intrinsic apoptotic pathways. Fisetin treated 2-D melanoma cultures displayed autophagic response concomitant with induction of apoptosis. Prolonged treatment (16days) with fisetin in a 3-D reconstituted melanoma model resulted in inhibition of melanoma progression with significant apoptosis, as evidenced by increased staining of cleaved Caspase-3 in the treated constructs. However, no difference in the expression of autophagic marker LC-3 was noted between treated and control groups. Fisetin treatment to 2-D melanoma cultures resulted in phosphorylation and activation of the multifunctional AMP-activated protein kinase (AMPK) involved in the regulation of diverse cellular processes, including autophagy and apoptosis. Silencing of AMPK failed to prevent cell death indicating that fisetin induced cytotoxicity is mediated through both AMPK-dependent and -independent mechanisms. Taken together, our studies confirm apoptosis as the primary mechanism through which fisetin inhibits melanoma cell growth and that activation of both extrinsic and intrinsic pathways contributes to fisetin induced cytotoxicity.

YB-1 expression promotes epithelial-to-mesenchymal transition in prostate cancer that is inhibited by a small molecule fisetin.

Epithelial-to-mesenchymal transition (EMT) plays an important role in prostate cancer (PCa) metastasis. The transcription/translation regulatory Y-box binding protein-1 (YB-1) is known to be associated with cancer metastasis. We observed that YB-1 expression increased with tumor grade and showed an inverse relationship with E-cadherin in a human PCa tissue array. Forced YB-1 expression induced a mesenchymal morphology that was associated with down regulation of epithelial markers. Silencing of YB-1 reversed mesenchymal features and decreased cell proliferation, migration and invasion in PCa cells. YB-1 is activated directly via Akt mediated phosphorylation at Ser102 within the cold shock domain (CSD). We next identified fisetin as an inhibitor of YB-1 activation. Computational docking and molecular dynamics suggested that fisetin binds on the residues from ß1 - ß4 strands of CSD, hindering Akt's interaction with YB-1. Calculated free binding energy ranged from -11.9845 to -9.6273 kcal/mol. Plasmon Surface Resonance studies showed that fisetin binds to YB-1 with an affinity of approximately 35 µM, with both slow association and dissociation. Fisetin also inhibited EGF induced YB-1 phosphorylation and markers of EMT both in vitro and in vivo. Collectively our data suggest that YB-1 induces EMT in PCa and identify fisetin as an inhibitor of its activation.

Fisetin inhibits human melanoma cell growth through direct binding to p70S6K and mTOR: findings from 3-D melanoma skin equivalents and computational modeling.

The incidence of melanoma continues to rise. Inspite of treatment advances, the prognosis remains grim once the disease has metastasized, emphasizing the need to explore additional therapeutic strategies. One such approach is through the use of mechanism-based dietary intervention. We previously showed that the flavonoid fisetin inhibits melanoma cell proliferation, in vitro and in vivo. Here, we studied fisetin-mediated regulation of kinases involved in melanoma growth and progression. Time-course analysis in 3-D melanoma constructs that transitioned from radial to vertical growth showed that fisetin treatment resulted in significant decrease in melanocytic lesions in contrast to untreated controls that showed large tumor nests and invading disseminated cells. Further studies in melanoma cultures and mouse xenografts showed that fisetin-mediated growth inhibition was associated with dephosphorylation of AKT, mTOR and p70S6K proteins. In silico modeling indicated direct interaction of fisetin with mTOR and p70S6K with favorable free energy values. These findings were validated by cell-free competition assays that established binding of fisetin to p70S6K and mTOR while little affinity was detected with AKT. Kinase activity studies reflected similar trend with % inhibition observed for p70S6K and mTOR at lower doses than AKT. Our studies characterized, for the first time, the differential interactions of any botanical agent with kinases involved in melanoma growth and demonstrate that fisetin inhibits mTOR and p70S6K through direct binding while the observed inhibitory effect of fisetin on AKT is mediated indirectly, through targeting interrelated pathways.

Oral infusion of pomegranate fruit extract inhibits prostate carcinogenesis in the TRAMP model.

We earlier provided evidence that oral consumption of pomegranate fruit extract (PFE) inhibits prostate cancer (PCa) cell growth in nude mice. To ascertain convincing evidence of chemopreventive effects of PFE against PCa, its efficacy requires to be evaluated in animal models that closely emulate human disease. Here, we provide evidence of remarkable tumor growth inhibitory effects of PFE using the TRAMP model. Mice received 0.1 and 0.2% PFE, equivalent to 250 and 500 ml of pomegranate juice, in drinking water, starting at 6 weeks and examined at 12, 20 and 34 weeks of age. In water-fed group, 100% mice developed palpable tumors by 20 weeks compared with only 30 and 20% in the 0.1 and 0.2% PFE-supplemented groups, respectively. At 34 weeks, palpable tumors were observed in 70 of 0.1% and only 50 of 0.2% PFE-supplemented mice. Compared with median survival of 43 weeks in water-fed mice, 0.1 and 0.2% PFE-supplemented mice exhibited median life expectancy of 73 and 92 weeks, respectively. Compared with respective water-fed groups, none of the mice in PFE-supplemented groups exhibited metastases to any of the distant organs at 20 weeks and only 20% mice exhibited metastasis at 34 weeks of age. Many of the PFE-supplemented animals had multiple foci of well-differentiated carcinoma but no evidence of poorly differentiated carcinoma. PFE supplementation resulted in simultaneous and significant inhibition of IGF-I/Akt/mTOR pathways in the prostate tissues and tumors. We suggest that pomegranate juice be evaluated in clinical trials in patients at high risk for developing PCa.