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.

Antimetastatic and Antitumor Activities of Orally Administered NAX014 Compound in a Murine Model of HER2-Positive Breast Cancer.

The natural isoquinoline alkaloid Berberine (BBR) has been shown to possess several therapeutic effects, including anticancer activity. Different BBR derivatives have been designed and synthesized in order to obtain new compounds with enhanced anticancer efficacy. We previously showed that intraperitoneal (IP) administration of the BBR-derived NAX014 compound was able to counteract HER-2 overexpressing mammary tumors onset and progression in transgenic mice. However, the IP administration was found to induce organ toxicity at doses higher than 2.5 mg/Kg. In this study, we evaluated the effect of intragastric (IG) administration of 20 mg/kg of NAX014 on both safety and anticancer efficacy in HER-2/neu transgenic mice. Furthermore, cancer cell dissemination and migration, tumor cell senescence and immunological changes were examined. Our results demonstrated that IG NAX014 administration delayed the onset of mammary tumors with no negative effects on health and survival. NAX014 reduced HER-2 overexpressing BC cells migration in vitro and the frequency of lung metastasis in HER-2/neu transgenic mice. A statistically significant increase of senescence-associated p16 expression was observed in tumors from NAX014-treated mice, and the induction of cell senescence was observed in HER-2 overexpressing BC cells after in vitro treatment with NAX014. Although NAX014 did not modulate the presence of tumor-infiltrating lymphocytes, the level of circulating TNF-α and VEGF was found to be reduced in NAX014-treated mice. The overall results address the NAX014 compound as potential tool for therapeutic strategies against HER-2 overexpressing breast cancer.

Potential application of berberine in the treatment of Escherichia coli sepsis.

Gram-negative sepsis ranks as the leading cause of death in intensive care units. Despite the development of new antibiotics, mortality from gram-negative sepsis remains high. The present study aims to investigate the in vivo effects of berberine (BBR) administration on septic death induced by intraperitoneal Escherichia coli injection. The results showed that (i) single 5 mg/kg dose of BBR increases the survival of septic mice, (ii) BBR administration improves the antimicrobial efficacy of antibiotic drug, (iii) BBR pre-treatment prevents improvements of BBR therapy without affecting the pro-survival effects of antibiotic drug. The effects of BBR administration were associated with immunological alterations represented by changes in CD4+ and CD8+ lymphocytes population and IL-6 and TNF-α production. This study highlighted the benefits of berberine administration as antibiotic adjuvant in E. coli sepsis. Furthermore, information about berberine-induced immunological perturbations and their influence on host response to infection and therapy has been shown.

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.

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.

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.

Berberine, an epiphany against cancer.

Alkaloids are used in traditional medicine for the treatment of many diseases. These compounds are synthesized in plants as secondary metabolites and have multiple effects on cellular metabolism. Among plant derivatives with biological properties, the isoquinoline quaternary alkaloid berberine possesses a broad range of therapeutic uses against several diseases. In recent years, berberine has been reported to inhibit cell proliferation and to be cytotoxic towards cancer cells. Based on this evidence, many derivatives have been synthesized to improve berberine efficiency and selectivity; the results so far obtained on human cancer cell lines support the idea that they could be promising agents for cancer treatment. The main properties of berberine and derivatives will be illustrated.

Modulation of the expression of folate cycle enzymes and polyamine metabolism by berberine in cisplatin-sensitive and -resistant human ovarian cancer cells.

Berberine is a natural isoquinoline alkaloid with significant antitumor activity against many types of cancer cells, including ovarian tumors. This study investigated the molecular mechanisms by which berberine differently affects cell growth of cisplatin (cDDP)-sensitive and -resistant and polyamine analogue cross-resistant human ovarian cancer cells. The results show that berberine suppresses the growth of cDDP-resistant cells more than the sensitive counterparts, by interfering with the expression of folate cycle enzymes, dihydrofolate reductase (DHFR) and thymidylate synthase (TS). In addition, the impairment of the folate cycle also seems partly ascribable to a reduced accumulation of folate, a vitamin which plays an essential role in the biosynthesis of nucleic acids and amino acids. This effect was observed in both lines, but especially in the resistant cells, correlating again with the reduced tolerance to this isoquinoline alkaloid. The data also indicate that berberine inhibits cellular growth by affecting polyamine metabolism, in particular through the upregulation of the key catabolic enzyme, spermidine/spermine N1-acetyltransferase (SSAT). In this regard, berberine is shown to stimulate the SSAT induction by the spermine analogue N1, N12 bisethylspermine (BESpm), which alone was also able to downregulate DHFR mRNA more than TS mRNA. We report that the sensitivity of resistant cells to cisplatin or to BESpm is reverted to the levels of sensitive cells by the co-treatment with berberine. These data confirm the intimate inter-relationships between folate cycle and polyamine pathways and suggest that this isoquinoline plant alkaloid could be a useful adjuvant therapeutic agent in the treatment of ovarian carcinoma.