Ewa Toton

Protein kinase Cε as a cancer marker and target for anticancer therapy.

Protein kinase Cε (PKCε) is a representative member of a family of novel PKC isoforms that are independent of calcium, but can be activated by phorbol esters, diacylglycerol (DAG) and phosphatidylserine (PS). This kinase is capable of modulating crucial cell functions, including proliferation, differentiation and survival. These activities depend on enzyme translocation to subcellular compartments upon binding DAG, PS or exogenous stimulators. PKCε initiates malignant transformation of cells through its effects on the Ras/Raf/MAPK pathway and displays the greatest carcinogenic potential of all PKC isoforms. PKCε also promotes tumor metastatic capacity and resistance to anti-cancer therapy. Overexpression of PKCε is found in numerous cancers including colon, breast, stomach, prostate, thyroid and lung and is considered an important marker of negative disease outcome. Although overexpression of PKCε is observed in tumors, it is not found in healthy tissues hence it has been suggested as a diagnostic marker or a putative target for specific inhibitors used for treatment of cancer. Research on selective inhibition of PKCε is under way and diverse approaches may become clinically applicable anti-tumor strategies. Suppression of the PKCε-encoding gene achieved through the antisense cDNA, suppression of PKCε with RNAi and inhibition achieved with translocation-inhibitory peptides may provide novel treatment strategies for cancer.

Signal transduction of constitutively active protein kinase C epsilon

The protein kinase C (PKC) family is the most prominent target of tumor-promoting phorbol esters. For the PKCepsilon isozyme, different intracellular localizations and oncogenic potential in several but not all experimental systems have been reported. To obtain information about PKCepsilon-signaling, we investigated the effects of constitutively active rat PKCepsilon (PKCepsilonA/E, alanine 159 is replaced by glutamic acid) in HeLa cells in a doxycycline-inducible vector. Upon induction of PKCepsilonA/E expression by doxycycline, the major part of PKCepsilonA/E was localized to the Golgi. This led (i) to phosphorylations of PKCepsilon(S729), Elk-1(S383), PDK1(S241) and Rb(S807/S811), (ii) to elevated expression of receptor of activated C kinase 2 (RACK2) after 12 h, and (iii) increased colony formation in soft agar, increased cell migration and invasion, but not to decreased doubling time. Following induction of PKCepsilonA/E-expression by doxycycline for 24 h and additional short-term treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), PKCepsilonA/E translocated to the plasma membrane and increased phosphorylation of MARCKS(S152/156). Treatment with doxycycline/TPA or TPA alone increased phosphorylations of Elk-1(S383), PDK1(S241), Rb(S807/S811), PKCdelta(T505), p38MAPK(T180/Y182), MEK1/2(S217/S221) and ERK2(T185/T187). MARCKS was not phosphorylated after treatment with TPA alone, demonstrating that in this system it is phosphorylated only by PKCepsilon localized to the plasma membrane but not by PKCalpha or delta, the other TPA-responsive PKC isozymes in HeLa cells. These results demonstrate that PKCepsilon can induce distinctly different signaling from the Golgi and from the plasma membrane.

Telomerase and drug resistance in cancer

It is well known that a decreased expression or inhibited activity of telomerase in cancer cells is accompanied by an increased sensitivity to some drugs (e.g., doxorubicin, cisplatin, or 5-fluorouracil). However, the mechanism of the resistance resulting from telomerase alteration remains elusive. There are theories claiming that it might be associated with telomere shortening, genome instability, hTERT translocation, mitochondria functioning modulation, or even alterations in ABC family gene expression. However, association of those mechanisms, i.e., drug resistance and telomerase alterations, is not fully understood yet. We review the current theories on the aspect of the role of telomerase in cancer cells resistance to therapy. We believe that revealing/unravelling this correlation might significantly contribute to an increased efficiency of cancer cells elimination, especially the most difficult ones, i.e., drug resistant.

Telomere shortening in Down syndrome patients--when does it start?

Down syndrome (DS) is one of the most common aneuploidy. In general population, its prevalence is 1:600-1:800 live births. It is caused by a trisomy of chromosome 21. DS is phenotypically manifested by premature aging, upward slant to the eyes, epicanthus, flattened face, and poor muscle tone. In addition to physical changes, this syndrome is characterized by early onset of diseases specific to old age, such as Alzheimers disease, vision and hearing problems, and precocious menopause. Since DS symptoms include premature aging, the shortening of telomeres might be one of the markers of cellular aging. Consequently, the aim of the study was to determine the length of the telomeres in leukocytes from the blood of juvenile patients with DS (n=68) compared to an age-matched control group (n=56) and also to determine the diagnostic or predictive value for this parameter. We show that, for the first time, in juveniles, the average relative telomere length in studied subjects is significantly longer than in the control group (50.46 vs. 40.56, respectively arbitrary units [AU]; p=0.0026). The results provide interesting basis for further research to determine the causes and consequences of telomere maintaining and the dynamics of this process in patients with DS.

Zapotin (5,6,2′,6′-tetramethoxyflavone) Modulates the Crosstalk Between Autophagy and Apoptosis Pathways in Cancer Cells with Overexpressed Constitutively Active PKCϵ

ABSTRACT Autophagy is important in the regulation of survival and death signaling pathways in cancer. PKCϵ revealed high transforming potential and the ability to increase cell migration, invasion, and metastasis. Zapotin (5,6,2′,6′-tetramethoxyflavone), a natural flavonoid, showed chemopreventive and anticancer properties. Previously, we reported that downmodulation of induced PKCϵ level by zapotin was associated with decreased migration and increased apoptosis in HeLa cell line containing doxycycline-inducible constitutively active PKCϵ (PKCϵA/E, Ala159 → Glu). Depending on the genetic and environmental content of cells, autophagy may either precede apoptosis or occur simultaneously. The purpose of this study was to assess the effect of zapotin on autophagy. Increasing concentration of zapotin (from 7.5 µM to 30 µM) caused an inhibition of the formation of autophagosomes and a decline in microtubule-associated protein 1 light chain 3 (LC3) protein levels. The gene expression level of major negative regulator of autophagy was noticeably increased. Moreover, the expression of the pivotal autophagy genes was decreased. These changes were accompanied by alternation in autophagy-related protein levels. In conclusion, our results implied that both the antiautophagic and the proapoptosis effect of zapotin in HeLaPKCϵA/E cells are associated with the protein kinase C epsilon signaling pathway and lead to programmed cell death.

The Synthetic Oleanane Triterpenoid HIMOXOL Induces Autophagy in Breast Cancer Cells via ERK1/2 MAPK Pathway and Beclin-1 Up-regulation

Autophagy is engaged in tumor growth and progression, but also acts as a cell death and tumor suppression initiator. Naturally-derived compounds and their derivatives constitute a rich source of autophagy modulators. This paper presents the study on the mechanism of action of oleanolic acid derivatives, HIMOXOL and Br-HIMOLID, in MCF7 breast cancer cells. Both compounds reduced MCF7 cell viability more efficiently than the parental compound. It is noteworthy that this effect was specific to MCF7 cancer cells, while in non-cancer MCF-12A cells the cytotoxicity of the studied compounds was significantly lower. Moreover, in contrast to oleanolic acid, the tested compounds were only able to increase autophagy in MCF7 cells. Interestingly, HIMOXOL caused a significantly (p<0.05) higher autophagy rate in MCF7 cells than Br-HIMOLID, as measured by an LC3 immuno-identification study. We also found that HIMOXOL upregulated Beclin-1 expression in MCF7 cells. The observed biological activity of the compound contributed to the modulation of the MAPK ERK1/2 pathway that is engaged in the regulation of autophagy signaling. Importantly, we revealed no proapoptotic activity of the compound in the studied cells. However, autophagy induction in MCF7 cancer cells was reflected in the significantly decreased viability of these cells. Thus, we conclude that HIMOXOL (but not Br-HIMOLID) might reveal a significant potential against breast cancer cells, since it might efficiently induce the main autophagy mediator and prognostic factor, BECN1.

The tetramethoxyflavone zapotin selectively activates protein kinase C epsilon, leading to its down-modulation accompanied by Bcl-2, c-Jun and c-Fos decrease.

Zapotin, a tetramethoxyflavone, is a natural compound with a wide spectrum of activities in neoplastic cells. Protein kinase C epsilon (PKCε) has been shown to be oncogenic, with the ability to increase cell migration, invasion and survival of tumor cells. Here we report that zapotin inhibits cell proliferation. In wild-type HeLa cells with basal endogenous expression of PKCε, the IC50 was found to be 17.9 ± 1.6 μM. In HeLa cells overexpressing doxycycline-inducible constitutively active PKCε (HeLaPKCεA/E), the IC50 was 7.6 ± 1.3 μM, suggesting that PKCε enhances the anti-proliferative effect of zapotin. Moreover, we found that zapotin selectively activated PKCε in comparison with other PKC family members, but attenuated doxycycline-induced PKCε expression. As a result of zapotin treatment for 6, 12 and 24 h, the doxycycline-induced levels of the two differently phosphorylated PKCε forms (87 kDa and 95 kDa) were decreased. Migration assays revealed that increasing concentrations of zapotin (from 3.5 to 15 μM) decreased migration of HeLaPKCεA/E cells. Furthermore, zapotin significantly increased the fraction of apoptotic cells in doxycycline-induced (HeLaPKCεA/E) cells after 24 h and decreased the levels of Bcl-2, c-Jun, c-Fos. This was accompanied by a degradation of PARP-1. In summary, activation of PKCε and down-modulation of the induced PKCε level by zapotin were associated with decreased migration and increased apoptosis. These observations are consistent with the previously reported chemopreventive and chemotherapeutic action of zapotin.

Autophagy as a Potential Therapeutic Target in Breast Cancer Treatment

One of the crucial reasons of breast cancer therapy failure is an impairment of mechanisms responsible for metabolism and cellular homeostasis, which makes it difficult to foresee the response to the treatment. Targeted therapy in breast cancer is dictated by the expression of specific molecules such as growth factor or hormone receptors. Many types of breast cancer exhibit different abnormalities in the apoptotic pathway, which confer the resistance to many forms of chemotherapy. Because of the fundamental importance of autophagy in the development and progression of cancer and its ability to affect treatment response, there has been an immense research on molecular regulation and signal transduction mechanisms that control this process. Here, we summarize the present knowledge concerning different breast cancer treatment strategies using drugs approved for the treatment of different breast cancer molecular subtypes with targeting pathways and factors associated with autophagy modulation/ regulation.

[Autophagy, new perspectives in anticancer therapy].

Autophagy, the process of degradation of unwanted or damaged cell elements, is extremely important for a variety of human diseases, especially cancers. This process influences various stages of initiation and progression of cancer, which is caused by overlapping signaling pathways of autophagy and carcinogenesis. However, due to the complexity of cancer as a systemic disease, the fate of tumor cells is not determined by one signal pathway. Chronic autophagy inhibition leads to tumor promotion, due to instability of the genome, defective cell growth, also as a result of cellular stress. However, increased induction of autophagy may be a mechanism for tumor cell survival in the state of hypoxia, acidosis, as well as under the influence of chemotherapy. Therefore, in the context of cancer development, the process of autophagy should be considered in two directions. Determination of the molecular mechanisms underlying the process of autophagy and its role in the carcinogenesis is a key element of the anticancer strategy. The main objective of modern oncology, which should eventually lead to personalized therapy, is the possibility to predict the response of a particular type of cancer to the used drug. Results of in vitro and in vivo studies show the magnitude of the relationship between changes in the genome, and response to the therapy. This information indicates the mechanism and thereby the target point of the drugs. In this review we focus on the mechanism of autophagy and its role in cancer therapy, which can help to understand the autophagy-cancer relationship and indicate the direction for the design of new drugs with anticancer activity.

Impact of PKCε downregulation on autophagy in glioblastoma cells

BackgroundSeveral efforts have been focused on identification of pathways involved in malignancy, progression, and response to treatment in Glioblastoma (GB). Overexpression of PKCε was detected in histological samples from GB, anaplastic astrocytoma, and gliosarcoma and is considered an important marker of negative disease outcome. In multiple studies on GB, autophagy has been shown as a survival mechanism during cellular stress, contributing to resistance against anti-cancer agents. The main object of this research was to determine the influence of PKCε downregulation on the expression of genes involved in autophagy pathways in glioblastoma cell lines U-138 MG and U-118 MG with high PKCε level.MethodsWe conducted siRNA-mediated knockdown of PKCε in glioblastoma cell lines and studied the effects of autophagy pathway. The expression of autophagy-related genes was analyzed using qPCR and Western blot analysis was carried out to assess protein levels. Immunostaining was used to detect functional autophagic maturation process.ResultsWe found that these cell lines exhibited a high basal expression of autophagy-related genes. Our results suggest that the loss of PKCε contributes to the downregulation of genes involved in autophagy pathways. Moreover, most of the changes we observed in Western blot analysis and endogenous immunofluorescence experiments confirmed dysfunction of autophagy programs. We found that knockdown of PKCε induced a decrease in the expression of Beclin1, Atg5, PI3K, whereas the expression of other autophagy-related proteins mTOR and Bcl2 was increased. Treatment of control siRNA glioma cells with rapamycin-induced autophagosome formation and increase in LC3-II level and caused a decrease in the expression of p62. Additionally, PKCε siRNA caused a diminution in the Akt phosphorylation at Ser473 and in the protein level in both cell lines. Moreover, we observed reduction in the adhesion of glioblastoma cells, accompanied by the decrease in total FAK protein level and phosphorylation.ConclusionsEffects of down-regulation of PKCε in glioma cells raised the possibility that the expression of PKCε is essential for the autophagic signal transduction pathways in these cells.Thus, our results identify an important role of PKCε in autophagy and may, more importantly, identifyit as a novel therapeutic target.