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.

Ectopic NGAL expression can alter sensitivity of breast cancer cells to EGFR, Bcl-2, CaM-K inhibitors and the plant natural product berberine.

Neutrophil gelatinase-associated lipocalin (NGAL, a.k.a Lnc2) is a member of the lipocalin family and has diverse roles. NGAL can stabilize matrix metalloproteinase-9 from autodegradation. NGAL is considered as a siderocalin that is important in the transport of iron. NGAL expression has also been associated with certain neoplasias and is implicated in the metastasis of breast cancer. In a previous study, we examined whether ectopic NGAL expression would alter the sensitivity of breast epithelial, breast and colorectal cancer cells to the effects of the chemotherapeutic drug doxorubicin. While abundant NGAL expression was detected in all the cells infected with a retrovirus encoding NGAL, this expression did not alter the sensitivity of these cells to doxorubicin as compared with empty vector-transduced cells. We were also interested in determining the effects of ectopic NGAL expression on the sensitivity to small-molecule inhibitors targeting key signaling molecules. Ectopic NGAL expression increased the sensitivity of MCF-7 breast cancer cells to EGFR, Bcl-2 and calmodulin kinase inhibitors as well as the natural plant product berberine. Furthermore, when suboptimal concentrations of certain inhibitors were combined with doxorubicin, a reduction in the doxorubicin IC 50 was frequently observed. An exception was observed when doxorubicin was combined with rapamycin, as doxorubicin suppressed the sensitivity of the NGAL-transduced MCF-7 cells to rapamycin when compared with the empty vector controls. In contrast, changes in the sensitivities of the NGAL-transduced HT-29 colorectal cancer cell line and the breast epithelial MCF-10A cell line were not detected compared with empty vector-transduced cells. Doxorubicin-resistant MCF-7/Dox (R) cells were examined in these experiments as a control drug-resistant line; it displayed increased sensitivity to EGFR and Bcl-2 inhibitors compared with empty vector transduced MCF-7 cells. These results indicate that NGAL expression can alter the sensitivity of certain cancer cells to small-molecule inhibitors, suggesting that patients whose tumors exhibit elevated NGAL expression or have become drug-resistant may display altered responses to certain small-molecule inhibitors.

Molecular pathways leading to oxidative stress-induced phosphorylation of Akt.

Oxidative stress can activate a variety of intracellular signaling pathways. The authors previously reported the CaM-K inhibitor KN-93 inhibited hydrogen peroxide-induced phosphorylation of Akt on threonine 308 (T308). In this report they demonstrate that phosphorylation of T308 in response to hydrogen peroxide treatment is not inhibited by LY294002, suggesting that phosphorylation of this residue in response to oxidative stress is largely PI3K independent. In contrast, hydrogen peroxide-induced phosphorylation of Akt on serine 473 (S473) was downregulated by both PI3K and CaM-K inhibition, indicating that hydrogen peroxideinduced phosphorylation of Akt on S473 was largely dependent on both PI3K and a CaM-K activity. Further, it is reported that p56(Lck) had a substantial role in hydrogen peroxide-induced phosphorylation of S473, but only a minimal role in hydrogen peroxide-induced phosphorylation of T308. These data suggest that in response to hydrogen peroxide, two pathways are activated in Jurkat T lymphocytes that converge to result in the phosphorylation of Akt on S473 and T308. One pathway involves the CaM-Ks that may directly phosphorylate Akt on T308. In this pathway, neither the Src kinases nor PI3K are required. The other pathway mediated by hydrogen peroxide results in the phosphorylation of Akt on S473 and requires CaM-K, PI3K, and Src activity.

Reactive oxygen species-induced activation of the MAP kinase signaling pathways.

An abundance of scientific literature exists demonstrating that oxidative stress influences the MAPK signaling pathways. This review summarizes these findings for the ERK, JNK, p38, and BMK1 pathways. For each of these different MAPK signaling pathways, the following is reviewed: the proteins involved in the signaling pathways, how oxidative stress can activate cellular signaling via these pathways, the types of oxidative stress that are known to induce activation of the different pathways, and the specific cell types in which oxidants induce MAPK responses. In addition, the functional outcome of oxidative stress-induced activation of these pathways is discussed. The purpose of this review is to provide the reader with an overall understanding and appreciation of oxidative stress-induced MAPK signaling.

Activation of the calcium/calmodulin-dependent protein kinases as a consequence of oxidative stress.

Oxygen radicals have diverse effects on cells. In many cases, exposure to reactive oxygen intermediates (ROI) can induce cell death. Conversely, there is also evidence that suggests oxygen radicals can activate signaling pathways that are thought to prevent cell death. In this review, the authors discuss the finding that hydrogen peroxide and ROI-generating treatments trigger the activation of the calcium/calmodulin-dependent kinases (CaM-kinases), and the potential role this activation has in preventing apoptosis. Evidence is presented that CaM-kinase activation occurs by both calcium dependent- and independent-pathways in response to ROIs. In addition, the idea is discussed that ROIs have the potential to lead to the phosphorylation of calmodulin and through this mechanism potentiate the activation of the CaM-kinases. The concept that inhibition of the CaM-kinases as a mechanism to sensitize cells to the damaging effects of ROIs is also presented. Contrasting these studies, evidence is presented that exposure of the CaM-kinases directly to hydrogen peroxide also has the apparent ability to inhibit their activity.

Inhibition of the CaM-kinases augments cell death in response to oxygen radicals and oxygen radical inducing cancer therapies in MCF-7 human breast cancer cells.

Many cancer treatments induce cell death through lethal oxidative stress. Oxidative stress also induces the activation of the calcium/calmodulin-dependent kinases (CaM-Ks), CaM-KII and CaM-KIV. In turn, the CaM-Ks are known to induce the activation of antiapoptotic signaling pathways, such as Akt, ERK, and NF-kappaB in many different cell types. The aim of this study was to determine the role of CaM-Kinases in resistance to hydrogen peroxide and three oxidative stress-inducing cancer therapies in MCF-7 breast cancer cells. We found that oxidative stress induced CaM-Kinase activity in MCF-7 breast cancer cells and that CaM-K inhibition increased hydrogen peroxide-induced cell death in MCF-7 human breast cancer cells. When MCF-7 cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy in the presence of a CaM-K inhibitor a greater level of cell killing was observed than when cells were treated with doxorubicin, ionizing radiation, or photodynamic therapy alone. In support of this finding, CaM-K inhibition increased hydrogen peroxide-induced apoptosis in MCF-7 cells, as determined by increased number of apoptotic cells, DNA fragmentation, and PARP cleavage. Pharmacological and molecular inhibition indicated that CaM-KII was participating in hydrogen peroxide-induced ERK phosphorylation in breast cancer cells indicating a potential mechanism by which this sensitization occurs. This is the first time that CaM-K inhibition is reported to sensitize cancer cells to reactive oxygen intermediate inducing cancer treatments.

OSU-03012 in the treatment of glioblastoma.

In an article presented in this issue of Molecular Pharmacology, Yacoub et al. (p. 589) examine the actions of 2-amino-N{4-5-(2-phenanthrenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-acetamide (OSU-03012) on both primary and glioblastoma cell lines. The authors found that OSU-03012 could induce tumor cell death by itself but also acted as a strong sensitizing agent to radiotherapy-induced cell death. Glioblastoma cells were also more sensitive to this compound than nontransformed astrocytes. Radiation-induced cell death was refractory to small interfering RNA-directed inhibition of PDK1 but not OSU-03012. These results indicate that OSU-03012, which has been thought to primarily mediate antitumor effects via the inhibition of PDK1, has actions independent of PDK1. Furthermore, the authors demonstrated that the effects of OSU-03012 were independent of ERB-B1-vIII and PTEN expression. These are important findings because they start to identify a new mechanism to sensitize glioblastoma cells and also suggest that OSU-03012 could be combined with existing inhibitors to further sensitize tumor cells. In glioblastoma cells, OSU-03012 seemed to induce apoptosis via endoplasmic reticulum stress-induced PERK-dependent signaling. OSU-03012-induced death of the glioblastoma was only weakly suppressed by the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp, suggesting that OSU-03012-induced cell death was largely caspase-independent. Overall, these are exciting results and suggest that new more effective treatment options may be obtainable for people suffering from these deadly tumors.

Calcium/calmodulin-dependent protein kinases as potential targets in cancer therapy.

In this review the authors discuss the expression and activation of a family of protein kinases known as the calcium/calmodulin-dependent kinases (CaM-kinase) and the role that these kinases have in the activation of antiapoptotic signalling pathways. In addition, the authors outline a novel mechanism of activation of these kinases by oxidative stress. Founded on this novel mechanism of activation and the role that these kinases have in activating antiapoptotic signalling pathways, the authors propose that the CaM-kinases would make very good targets for sensitising cancer cells to certain therapeutic treatments. Furthermore, the authors discuss the role that these kinases have in cell transformation and in the regulation of the cell cycle. Based on these roles the authors suggest that inhibition of the CaM-kinases not only has the potential to sensitise cancer cells, but also has the potential to induce cytostasis in these cells.

Calcium/calmodulin-dependent kinase I and calcium/calmodulin-dependent kinase kinase participate in the control of cell cycle progression in MCF-7 human breast cancer cells.

Calcium is universally required for cell growth and proliferation. Calmodulin is the main intracellular receptor for calcium. Although calcium and calmodulin are well known to be required for cell cycle regulation, the target pathways for their action remain poorly defined. Potential targets include the calcium/calmodulin-dependent kinases (CaM-K). The aim of this study was to determine the role of the CaM-Ks on cell proliferation and progress through the cell cycle in breast cancer cells. CaM-KI inhibition with either KN-93 or specific interfering RNA (siRNA) caused an arrest in the cell cycle in the human breast cancer cell line, MCF-7. This arrest occurred in the G(1) phase of the cell cycle. Supporting this finding, CaM-K inhibition using KN-93 also resulted in a reduction of cyclin D1 protein and pRb phosphorylation when cells were compared with control cultures. Furthermore, inhibition of the upstream activator of CaM-KI, CaM-KK, using siRNA also resulted in cell cycle arrest. In summary, CaM-KK and CaM-KI participate in the control of the G(0)-G(1) restriction check point of the cell cycle in human breast cancer cells. This arrest seems due to an inhibition in cyclin D1 synthesis and a reduction in pRb phosphorylation. To the best of our knowledge, this is the first time that CaM-KK has been reported to be involved in mammalian cell cycle regulation and that CaM-Ks are regulating breast cancer cell cycle.

Inhibition of CREB transcriptional activity in human T lymphocytes by oxidative stress.

Hydrogen peroxide (HP) induced the phosphorylation of cAMP response element binding protein (CREB) on Ser133 in Jurkat T lymphocytes via p38 and MSK1. Although CREB Ser133 was phosphorylated, increases in HP-stimulated CREB-mediated transcription were absent. T lymphocyte stimulation with anti-CD3 and anti-CD28 induced CREB Ser133 phosphorylation, as well as CREB-mediated transcriptional activity. When CD3/CD28-stimulated lymphocytes were treated with HP, Ser133 was phosphorylated, but TCR-induced CREB-mediated transcriptional activity was reduced. These data provide insight into a potential mechanism by which oxidative stress can alter T cell receptor-induced CREB activation and responsiveness.

Redox regulation of the calcium/calmodulin-dependent protein kinases.

Reactive oxygen intermediates (ROI) have been viewed traditionally as damaging to the cell. However, a predominance of evidence has shown that ROI can also function as important activators of key cellular processes, and ROI have been shown to play a vital role in cell signaling networks. The calcium/calmodulin-dependent protein kinases (CaM kinases) are a family of related kinases that are activated in response to increased intracellular calcium concentrations. In this report we demonstrate that hydrogen peroxide treatment results in the activation of both CaM kinase II and IV in Jurkat T lymphocytes. Surprisingly, this activation occurs in the absence of any detectable calcium flux, suggesting a novel means for the activation of these kinases. Treatment of Jurkat cells with phorbol 12-myristate 13-acetate (PMA), which does not cause a calcium flux, also activated the CaM kinases. The addition of catalase to the cultures inhibited PMA-induced activation of the CaM kinases, suggesting that similar to hydrogen peroxide, PMA also activates the CaM kinases via the production of ROI. One mechanism by which this likely occurs is through oxidation and consequential inactivation of cellular phosphatases. In support of this concept, okadaic acid and microcystin-LR, which are inhibitors of protein phosphatase 2A (PP2A), induced CaM kinase II and IV activity in these cells. Overall, these results demonstrate a novel mechanism by which ROI can induce CaM kinase activation in T lymphocytes.

Models of anergy in the human Jurkat T cell line.

We investigated two model systems to study anergy in a human T cell line. OKT3 or calcium ionophore stimulation of Jurkat cells, in the absence of costimulation, resulted in a steep reduction in the transcription and secretion of IL-2 in response to subsequent stimulation via CD3 and CD28. Treatment of anergic Jurkat cells with the combination of the phorbol ester, PMA, and ionomycin restored IL-2 production in cells rendered anergic by both mechanisms. However, hydrogen peroxide, which also stimulates kinases downstream of the proposed block that occurs in anergic murine cells, did not reverse the anergic state of these cells induced by either stimulus. The cause of unresponsiveness in these two models was found to differ. OKT3-induced anergy resulted in a substantial down-regulation of the CD3 on these cells. In contrast, anergy induced by treatment with a calcium ionophore did not result in CD3 down-regulation. These data indicate that the Jurkat cell line may serve as a suitable model for studying anergy in human T cells; however, the mechanism by which anergy is induced may vary dramatically in response to these two commonly used anergy-inducing strategies. Understanding the similarities and differences between these two models of anergy may lead to a better overall understanding of the anergic state of the T cell.

Participation of the calcium/calmodulin-dependent kinases in hydrogen peroxide-induced Ikappa B phosphorylation in human T lymphocytes.

NF-kappaB is an important transcription factor that has a role in a variety of responses such as inflammation, oncogenesis, apoptosis, and viral replication. Oxidative stress is well known to induce the activation of NF-kappaB. Cells can be exposed to either endogenously produced oxidants or oxidants produced by surrounding cells. In addition, ischemia reperfusion and certain cancer therapies such as chemotherapy and photodynamic therapy are thought to result in oxygen radical production. Because of the important role that NF-kappaB has in multiple responses, it is critical to determine the mechanisms by which oxidative stress induces NF-kappaB activity. We report that the calmodulin antagonist W-7 and the calcium/calmodulin-dependent (CaM) kinase inhibitors KN-93 and K252a, can block oxidative stress-induced IkappaB phosphorylation in Jurkat T lymphocytes. Furthermore, KN-93 but not KN-92 can block hydrogen peroxide-induced Akt and IKK phosphorylation. In addition, we found that expression of a kinase-dead CaM-KIV construct in two cell lines inhibits IkappaB phosphorylation or degradation and that expression of CaM-KIV augments hydrogen peroxide-induced IkappaB phosphorylation and degradation. Although the CaM kinases appear to be required for this response, increases in intracellular calcium do not appear to be required. These results identify the CaM kinases as potential targets that can be used to minimize NF-kappaB activation in response to oxidative stress.