Wild type and gain of function mutant TP53 can regulate the sensitivity of pancreatic cancer cells to chemotherapeutic drugs, EGFR/Ras/Raf/MEK, and PI3K/mTORC1/GSK-3 pathway inhibitors, nutraceuticals and alter metabolic properties.

TP53 is a master regulator of many signaling and apoptotic pathways involved in: aging, cell cycle progression, gene regulation, growth, apoptosis, cellular senescence, DNA repair, drug resistance, malignant transformation, metastasis, and metabolism. Most pancreatic cancers are classified as pancreatic ductal adenocarcinomas (PDAC). The tumor suppressor gene TP53 is mutated frequently (50-75%) in PDAC. Different types of TP53 mutations have been observed including gain of function (GOF) point mutations and various deletions of the TP53 gene resulting in lack of the protein expression. Most PDACs have point mutations at the KRAS gene which result in constitutive activation of KRas and multiple downstream signaling pathways. It has been difficult to develop specific KRas inhibitors and/or methods that result in recovery of functional TP53 activity. To further elucidate the roles of TP53 in drug-resistance of pancreatic cancer cells, we introduced wild-type (WT) TP53 or a control vector into two different PDAC cell lines. Introduction of WT-TP53 increased the sensitivity of the cells to multiple chemotherapeutic drugs, signal transduction inhibitors, drugs and nutraceuticals and influenced key metabolic properties of the cells. Therefore, TP53 is a key molecule which is critical in drug sensitivity and metabolism of PDAC.

Effects of the MDM2 inhibitor Nutlin-3a on sensitivity of pancreatic cancer cells to berberine and modified berberines in the presence and absence of WT-TP53.

Approaches to improve pancreatic cancer therapy are essential as this disease has a very bleak outcome. Approximately 80% of pancreatic cancers are pancreatic ductal adenocarcinomas (PDAC). A key regulatory gene frequently mutated (∼75%) in PDAC is the TP53 tumor suppressor gene which controls the transcription of multiple genes involved in cell cycle progression, apoptosis, cancer progression and other growth regulatory processes. The mouse double minute 2 homolog (MDM2) gene product is a nuclear-localized E3 ubiquitin ligase and negatively regulates the TP53 protein which results in its proteasomal degradation. Various MDM2 inhibitors have been isolated and examined in clinical trials, especially in patients with hematological malignancies. Nutlin-3a is one of the first MDM2 inhibitors isolated. Berberine (BBR) is a natural product found in many fruits and berries and used in traditional medicine for centuries. It has many biological effects, and some are anti-proliferative in nature. BBR may activate the expression of TP53 and inhibit cell cycle progression as well as other events important in cell growth. To understand more about the potential of compounds like BBR and chemical modified BBRs (NAX compounds) to sensitize PDAC cells to MDM2 inhibitors, we introduced either WT-TP53 or the pLXSN empty vector control into two PDAC cell lines, one lacking expression of TP53 (PANC-28) and one with gain-of-function mutant TP53 on both alleles (MIA-PaCa-2). Our results indicate that nutlin-3a was able to increase the sensitivity to BBR and certain NAX compounds. The effects of nutlin-3a were usually more substantial in those cells containing an introduced WT TP53 gene. These results highlight the importance of knowledge of the type of TP53 mutation that is present in cancer patients before the administration of drugs which function by stabilization of the TP53 protein.

The Combination of AHCC and ETAS Decreases Migration of Colorectal Cancer Cells, and Reduces the Expression of LGR5 and Notch1 Genes in Cancer Stem Cells: A Novel Potential Approach for Integrative Medicine.

The AHCC standardized extract of cultured Lentinula edodes mycelia, and the standardized extract of Asparagus officinalis stem, trademarked as ETAS, are well known supplements with immunomodulatory and anticancer potential. Several reports have described their therapeutic effects, including antioxidant and anticancer activity and improvement of immune response. In this study we aimed at investigating the effects of a combination of AHCC and ETAS on colorectal cancer cells and biopsies from healthy donors to assess the possible use in patients with colorectal cancer. Our results showed that the combination of AHCC and ETAS was synergistic in inducing a significant decrease in cancer cell growth, compared with single agents. Moreover, the combined treatment induced a significant increase in apoptosis, sparing colonocytes from healthy donors, and was able to induce a strong reduction in migration potential, accompanied by a significant modulation of proteins involved in invasiveness. Finally, combined treatment was able to significantly downregulate LGR5 and Notch1 in SW620 cancer stem cell (CSC) colonospheres. Overall, these findings support the potential therapeutic benefits of the AHCC and ETAS combinatorial treatment for patients with colorectal cancer.

GSK-3ß Can Regulate the Sensitivity of MIA-PaCa-2 Pancreatic and MCF-7 Breast Cancer Cells to Chemotherapeutic Drugs, Targeted Therapeutics and Nutraceuticals.

Glycogen synthase kinase-3 (GSK-3) is a regulator of signaling pathways. KRas is frequently mutated in pancreatic cancers. The growth of certain pancreatic cancers is KRas-dependent and can be suppressed by GSK-3 inhibitors, documenting a link between KRas and GSK-3. To further elucidate the roles of GSK-3ß in drug-resistance, we transfected KRas-dependent MIA-PaCa-2 pancreatic cells with wild-type (WT) and kinase-dead (KD) forms of GSK-3ß. Transfection of MIA-PaCa-2 cells with WT-GSK-3ß increased their resistance to various chemotherapeutic drugs and certain small molecule inhibitors. Transfection of cells with KD-GSK-3ß often increased therapeutic sensitivity. An exception was observed with cells transfected with WT-GSK-3ß and sensitivity to the BCL2/BCLXL ABT737 inhibitor. WT-GSK-3ß reduced glycolytic capacity of the cells but did not affect the basal glycolysis and mitochondrial respiration. KD-GSK-3ß decreased both basal glycolysis and glycolytic capacity and reduced mitochondrial respiration in MIA-PaCa-2 cells. As a comparison, the effects of GSK-3 on MCF-7 breast cancer cells, which have mutant PIK3CA, were examined. KD-GSK-3ß increased the resistance of MCF-7 cells to chemotherapeutic drugs and certain signal transduction inhibitors. Thus, altering the levels of GSK-3ß can have dramatic effects on sensitivity to drugs and signal transduction inhibitors which may be influenced by the background of the tumor.

Sensitivity of pancreatic cancer cells to chemotherapeutic drugs, signal transduction inhibitors and nutraceuticals can be regulated by WT-TP53.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic malignancy. Approximately 85% of pancreatic cancers are classified as PDACs. The survival of PDAC patients is very poor and only 5-10% of patients survive 5 years after diagnosis. Mutations at the KRAS and TP53 gene are frequently observed in PDAC patients. The PANC-28 cell line lacks wild-type (WT) TP53. In the following study, we have investigated the effects of restoration of WT TP53 activity on the sensitivity of PANC-28 pancreatic cancer cells to various drugs which are used to treat PDAC patients as well as other cancer patients. In addition, we have examined the effects of signal transduction inhibitors which target critical pathways frequently deregulated in cancer. The effects of the anti-diabetes drug metformin and the anti-malarial drug chloroquine were also examined as these drugs may be repurposed to treat other diseases. Finally, the effects of certain nutraceuticals which are used to treat various ailments were also examined. Introduction of WT-TP53 activity in PANC-28 PDAC cells, can increase their sensitivity to various drugs. Attempts are being made clinically to increase TP53 activity in various cancer types which will often inhibit cell growth by multiple mechanisms.

Abilities of ß-Estradiol to interact with chemotherapeutic drugs, signal transduction inhibitors and nutraceuticals and alter the proliferation of pancreatic cancer cells.

Improving the effects of chemotherapy and reducing the side effects are important goals in cancer research. Various approaches have been examined to enhance the effectiveness of chemotherapy. For example, signal transduction inhibitors or hormonal based approaches have been included with chemo- or radio-therapy. MIA-PaCa-2 and BxPC-3 pancreatic ductal adenocarcinoma (PDAC) cells both express the estrogen receptor (ER). The effects of ß-estradiol on the growth of PDAC cells has not been examined yet the ER is expressed in PDAC cells. We have examined the effects of combining ß-estradiol with chemotherapeutic drugs, signal transcription inhibitors, natural products and nutraceuticals on PDAC. In most cases, inclusion of ß-estradiol with chemotherapeutic drugs increased chemosensitivity. These results indicate some approaches involving ß-estradiol which may be used to increase the effectiveness of chemotherapeutic and other drugs on the growth of PDAC.

Abilities of berberine and chemically modified berberines to interact with metformin and inhibit proliferation of pancreatic cancer cells.

Pancreatic cancer is devastating cancer worldwide with few if any truly effective therapies. Pancreatic cancer has an increasing incidence and may become the second leading cause of death from cancer. Novel, more effective therapeutic approaches are needed as pancreatic cancer patients usually survive for less than a year after being diagnosed. Control of blood sugar levels by the prescription drug metformin in diseases such as diabetes mellitus has been examined in association with pancreatic cancer. While the clinical trials remain inconclusive, there is hope that certain diets and medications may affect positively the outcomes of patients with pancreatic and other cancers. Other natural compounds may share some of the effects of metformin. One "medicinal" fruit consumed by millions worldwide is berberine (BBR). Metformin and BBR both activate AMP-activated protein kinase (AMPK) which is a key mediator of glucose metabolism. Glucose metabolism has been shown to be very important in cancer and its significance is increasing. In the following studies, we have examined the effects of metformin, BBR and a panel of modified BBRs (NAX compounds) and chemotherapeutic drugs on the growth of four different human pancreatic adenocarcinoma cell lines (PDAC). Interestingly, the effects of metformin could be enhanced by BBR and certain modified BBRs. Upon restoration of WT-TP53 activity in MIA-PaCa-2 cells, an altered sensitivity to the combination of certain NAX compounds and metformin was observed compared to the parental cells which normally lack WT-TP53. Certain NAX compounds may interact with WT-TP53 and metformin treatment to alter the expression of key molecules involved in cell growth. These results suggest a therapeutic approach by combining certain pharmaceutical drugs and nutraceuticals to suppress the growth of cancer cells.

Effects of the MDM-2 inhibitor Nutlin-3a on PDAC cells containing and lacking WT-TP53 on sensitivity to chemotherapy, signal transduction inhibitors and nutraceuticals.

Mutations at the TP53 gene are readily detected (approximately 50-75%) in pancreatic ductal adenocarcinoma (PDAC) patients. TP53 was previously thought to be a difficult target as it is often mutated, deleted or inactivated on both chromosomes in certain cancers. In the following study, the effects of restoration of wild-type (WT) TP53 activity on the sensitivities of MIA-PaCa-2 pancreatic cancer cells to the MDM2 inhibitor nutlin-3a in combination with chemotherapy, targeted therapy, as well as, nutraceuticals were examined. Upon introduction of the WT-TP53 gene into MIA-PaCa-2 cells, which contain a TP53 gain of function (GOF) mutation, the sensitivity to the MDM2 inhibitor increased. However, effects of nutlin-3a were also observed in MIA-PaCa-2 cells lacking WT-TP53, as upon co-treatment with nutlin-3a, the sensitivity to certain inhibitors, chemotherapeutic drugs and nutraceuticals increased. Interestingly, co-treatment with nutlin-3a and certain chemotherapeutic drug such as irinotecan and oxaliplatin resulted in antagonistic effects in cells both lacking and containing WT-TP53 activity. These studies indicate the sensitizing abilities that WT-TP53 activity can have in PDAC cells which normally lack WT-TP53, as well as, the effects that the MDM2 inhibitor nutlin-3a can have in both cells containing and lacking WT-TP53 to various therapeutic agents.

Introduction of WT-TP53 into pancreatic cancer cells alters sensitivity to chemotherapeutic drugs, targeted therapeutics and nutraceuticals.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, highly metastatic malignancy and accounts for 85% of pancreatic cancers. PDAC patients have poor prognosis with a five-year survival of only 5-10%. Mutations at the TP53 gene are readily detected in pancreatic tumors isolated from PDAC patients. We have investigated the effects of restoration of wild-type (WT) TP53 activity on the sensitivity of pancreatic cancer cells to: chemotherapy, targeted therapy, as well as, nutraceuticals. Upon introduction of the WT-TP53 gene into the MIA-PaCa-2 pancreatic cancer cell line, the sensitivity to drugs used to treat pancreatic cancer cells such as: gemcitabine, fluorouracil (5FU), cisplatin, irinotecan, oxaliplatin, and paclitaxel increased significantly. Likewise, the sensitivity to drugs used to treat other cancers such as: doxorubicin, mitoxantrone, and 4 hydroxy tamoxifen (4HT) also increased upon introduction of WT-TP53 into MIA-PaCa-2 cells. Furthermore, the sensitivity to certain inhibitors which target: PI3K/mTORC1, PDK1, SRC, GSK-3, and biochemical processes such as proteasomal degradation and the nutraceutical berberine as increased upon introduction of WT-TP53. Furthermore, in some cases, cells with WT-TP53 were more sensitive to the combination of drugs and suboptimal doses of the MDM2 inhibitor nutlin-3a. However, TP53-independent effects of nutlin-3a were observed upon treatment with either a proteasomal or a PI3K/mTOR inhibitor. These studies indicate the sensitizing effects that WT-TP53 can have in PDAC cells which normally lack WT-TP53 to various therapeutic agents and suggest approaches to improve PDAC therapy.

Oxidative stress: role of physical exercise and antioxidant nutraceuticals in adulthood and aging.

Physical exercise is considered to be one of the beneficial factors of a proper lifestyle and is nowadays seen as an indispensable element for good health, able to lower the risk of disorders of the cardiovascular, endocrine and osteomuscular apparatus, immune system diseases and the onset of potential neoplasms. A moderate and programmed physical exercise has often been reported to be therapeutic both in the adulthood and in aging, since capable to promote fitness. Regular exercise alleviates the negative effects caused by free radicals and offers many health benefits, including reduced risk of all-cause mortality, sarcopenia in the skeletal muscle, chronic disease, and premature death in elderly people. However, physical performance is also known to induce oxidative stress, inflammation, and muscle fatigue. Many efforts have been carried out to identify micronutrients and natural compounds, also known as nutraceuticals, able to prevent or attenuate the exercise-induced oxidative stress and inflammation. The aim of this review is to discuss the benefits deriving from a constant physical activity and by the intake of antioxidant compounds to protect the body from oxidative stress. The attention will be focused mainly on three natural antioxidants, which are quercetin, resveratrol and curcumin. Their properties and activity will be described, as well as their benefits on physical activity and on aging, which is expected to increase through the years and can get favorable benefits from a constant exercise activity.

Metformin influences drug sensitivity in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive, highly metastatic malignancy and accounts for 85% of pancreatic cancers. PDAC patients have poor prognosis with a five-year survival of only 5-10% after diagnosis and treatment. Pancreatic cancer has been associated with type II diabetes as the frequency of recently diagnosed diabetics that develop pancreatic cancer within a 10-year period of initial diagnosis of diabetes in increased in comparison to non-diabetic patients. Metformin is a very frequently prescribed drug used to treat type II diabetes. Metformin acts in part by stimulating AMP-kinase (AMPK) and results in the suppression of mTORC1 activity and the induction of autophagy. In the following studies, we have examined the effects of metformin in the presence of various chemotherapeutic drugs, signal transduction inhibitors and natural products on the growth of three different PDAC lines. Metformin, by itself, was not effective at suppressing growth of the pancreatic cancer cell lines at concentration less than 1000 nM, however, in certain PDAC lines, a suboptimal dose of metformin (250 nM) potentiated the effects of various chemotherapeutic drugs used to treat pancreatic cancer (e.g., gemcitabine, cisplatin, 5-fluorouracil) and other cancer types (e.g., doxorubicin, docetaxel). Furthermore, metformin could increase anti-proliferative effects of mTORC1 and PI3K/mTOR inhibitors as well as natural products such as berberine and the anti-malarial drug chloroquine in certain PDAC lines. Thus, metformin can enhance the effects of certain drugs and signal transduction inhibitors which are used to treat pancreatic and various other cancers.

Effects of berberine, curcumin, resveratrol alone and in combination with chemotherapeutic drugs and signal transduction inhibitors on cancer cells-Power of nutraceuticals.

Over the past fifty years, society has become aware of the importance of a healthy diet in terms of human fitness and longevity. More recently, the concept of the beneficial effects of certain components of our diet and other compounds, that are consumed often by different cultures in various parts of the world, has become apparent. These "healthy" components of our diet are often referred to as nutraceuticals and they can prevent/suppress: aging, bacterial, fungal and viral infections, diabetes, inflammation, metabolic disorders and cardiovascular diseases and have other health-enhancing effects. Moreover, they are now often being investigated because of their anti-cancer properties/potentials. Understanding the effects of various natural products on cancer cells may enhance their usage as anti-proliferative agents which may be beneficial for many health problems. In this manuscript, we discuss and demonstrate how certain nutraceuticals may enhance other anti-cancer drugs to suppress proliferation of cancer cells.

Regulation of GSK-3 activity by curcumin, berberine and resveratrol: Potential effects on multiple diseases.

Natural products or nutraceuticals promote anti-aging, anti-cancer and other health-enhancing effects. A key target of the effects of natural products may be the regulation of the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway. This review will focus on the effects of curcumin (CUR), berberine (BBR) and resveratrol (RES), on the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway, with a special focus on GSK-3. These natural products may regulate the pathway by multiple mechanisms including: reactive oxygen species (ROS), cytokine receptors, mirco-RNAs (miRs) and many others. CUR is present the root of turmeric (Curcuma longa). CUR is used in the treatment of many disorders, especially in those involving inflammatory processes which may contribute to abnormal proliferation and promote cancer growth. BBR is also isolated from various plants (Berberis coptis and others) and is used in traditional medicine to treat multiple diseases/conditions including: diabetes, hyperlipidemia, cancer and bacterial infections. RES is present in red grapes, other fruits and berries such as blueberries and raspberries. RES may have some anti-diabetic and anti-cancer effects. Understanding the effects of these natural products on the PI3K/PTEN/Akt/mTORC1/GSK-3 pathway may enhance their usage as anti-proliferative agent which may be beneficial for many health problems.

Effects of resveratrol, curcumin, berberine and other nutraceuticals on aging, cancer development, cancer stem cells and microRNAs.

Natural products or nutraceuticals have been shown to elicit anti-aging, anti-cancer and other health-enhancing effects. A key target of the effects of natural products may be the regulation of microRNA (miR) expression which results in cell death or prevents aging, diabetes, cardiovascular and other diseases. This review will focus on a few natural products, especially on resveratrol (RES), curcumin (CUR) and berberine (BBR). RES is obtained from the skins of grapes and other fruits and berries. RES may extend human lifespan by activating the sirtuins and SIRT1 molecules. CUR is isolated from the root of turmeric (Curcuma longa). CUR is currently used in the treatment of many disorders, especially in those involving an inflammatory process. CUR and modified derivatives have been shown to have potent anti-cancer effects, especially on cancer stem cells (CSC). BBR is also isolated from various plants (e.g., Coptis chinensis) and has been used for centuries in traditional medicine to treat diseases such as adult- onset diabetes. Understanding the benefits of these and other nutraceuticals may result in approaches to improve human health.

Targeting the PI3K/AKT/mTOR signaling network in acute myelogenous leukemia.

BACKGROUND: The PI3K/Akt/mammalian target of rapamycin (mTOR) signaling pathway plays a central role in cell growth, proliferation and survival not only under physiological conditions but also in a variety of tumor cells. Therefore, the PI3K/Akt/mTOR axis may be a critical target for cancer therapy. OBJECTIVE: This review discusses how PI3K/Akt/mTOR signaling network is constitutively active in acute myelogenous leukemia (AML), where it strongly influences proliferation, survival and drug-resistance of leukemic cells, and how effective targeting of this pathway with pharmacological inhibitors, used alone or in combination with existing drugs, may result in suppression of leukemic cell growth, including leukemic stem cells. METHODS: We searched the literature for articles dealing with activation of this pathway in AML and highlighting the efficacy of small molecules directed against the PI3K/Akt/mTOR signaling cascade. CONCLUSIONS: The limit of acceptable toxicity for standard chemotherapy has been reached in AML. Therefore, new therapeutic strategies are needed. Targeting the PI3K/Akt/mTOR signaling network with small molecule inhibitors, alone or in combinations with other drugs, may result in less toxic and more efficacious treatment of AML patients. Efforts to exploit selective inhibitors of the PI3K/Akt/mTOR pathway that show effectiveness and safety in the clinical setting are currently underway.

Constitutive traffic of melanocortin-4 receptor in Neuro2A cells and immortalized hypothalamic neurons.

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds alpha-melanocyte-stimulating hormone (alpha-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of alpha-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toalpha-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that alpha-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.

Caspase-9 is the upstream caspase activated by 8-methoxypsoralen and ultraviolet-A radiation treatment of Jurkat T leukemia cells and normal T lymphocytes.

BACKGROUND AND OBJECTIVES: A combination of 8-methoxypsoralen and ultraviolet-A radiation (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however scarce information is available concerning the apoptotic pathways activated by PUVA. DESIGN AND METHODS: We used Jurkat human T leukemia cells and normal T lymphocytes to analyze the PUVA-triggered caspase activation pattern by means of immunoblot analysis, in vitro caspase activity assays, and selective caspase inhibitors coupled to flow cytometric analysis. RESULTS: PUVA treatment induced activation of apical caspases-9 and -8, and of effector caspases-3 and -7 in Jurkat cells and human T lymphocytes. While activation of caspase-9 occurred as early as 1 h after PUVA treatment of Jurkat cells, procaspase-8 cleavage was delayed and was detected 6 h after the exposure. Also in normal T lymphocytes, cleavage of caspase-8 was subsequent to activation of caspase-9. PUVA-dependent proteolytic cleavage of procaspase-8 was blocked by inhibitors selective for either caspase-9 or -3. Moreover, procaspase-8 was cleaved in vitro by activated caspase-3, which gave rise to proteolytic fragments equivalent to those generated in vivo. INTERPRETATION AND CONCLUSIONS: Activation of caspase-8 in PUVA-treated Jurkat cells and normal T lymphocytes is secondary to up-regulation of caspase-9. Overall, our results identify caspase-9 as the critical upstream caspase initiating apoptosis by PUVA in Jurkat T-cells and human T lymphocytes.