Proapoptotic and proautophagic activity of 20-hydroxyecdysone in breast cancer cells in vitro.

The present study was designed to identify the biological activity of three ecdysones, i.e., 20-hydroxyecdysone (20-HE), ajugasterone C, and polypodine B isolated from Serratula coronata. The main objective was to investigate the molecular mechanism of the biological activity of those compounds and to assess their impact on breast cancer cell survival and cell cycle. Cell lines were selected according to their hormone receptor status since this factor is perceived as a crucial one in the cancer prognosis as well as cancer cell response to therapy. Consequently, MCF7 (ER/PR+, HER2-), T-47D (ER/PR+, HER2-/+), and MDA-MB-231 (ER/PR-, HER2-) were enrolled in the study. Additionally, a non-tumorigenic, MCF10A cells were selected to verify any potential specificity to cancer cells. Interestingly, none of the studied compounds affected the viability of MCF10A cells while cancer cells were altered, albeit in different ways. Polypodine B did not affect the viability or cell cycle distribution of studied breast cancer cells. By contrast, 20-HE and ajugasterone C significantly inhibited the viability of triple-negative cell line, MDA-MB-231. Interestingly, 20-HE revealed proapoptotic activity in MDA-MB-231 and T-47D cells that was manifested by alterations in PARP, Bax, and Bcl-2 levels as well as caspase-3 activation. Moreover, 20-HE induced autophagy that was mediated by modification of autophagy-associated proteins, i.e., LC3, p62, and mTOR, but only in MDA-MB-231 cells. This study is the first to report diverse biological activity of phytoecdysones in different breast cancer cells, that suggests association with molecular characteristics including receptor status but also other biological properties and genetic markers.

Effect of 3-O-acetylaleuritolic acid from in vitro-cultured Drosera spatulata on cancer cells survival and migration.

BACKGROUND: Drosera spatulata is a source of many compounds such as naphthoquinones, phenolic acids, flavonoids, anthocyanins, and naphthalene derivatives. Unfortunately, the information regarding the biological activity and chemical profile of those compounds is still incomplete. Herein, we investigated the biological activity of 3-O-acetylaleuritolic acid (3-O-AAA) in cancer cell lines. METHODS: The cell viability of HeLa, HT-29, MCF7, and MCF12A cells was assessed using MTT assay. Proliferation potential was assessed using the clonogenic assay and flow cytometry. Migration modulation was tested using a scratch assay. Protein expression was analyzed by immunoblotting. RESULTS: 3-O-AAA significantly inhibited the growth of all tested tumor cells. The results of the colony formation assay suggested cytostatic properties of the studied compound. The scratch assay showed that 3-O-AAA was an efficient migration inhibitor in a dose-dependent manner. Moreover, it caused modulation of mTOR, beclin1, and Atg5 proteins suggesting a possible role of the compound in autophagy induction. CONCLUSION: Collectively, these results demonstrated that 3-O-AAA inhibited the proliferation and migration of cancer cell lines as well as contributed to autophagy induction showing some anticancer properties.

Comparison of bioactive compounds content in leaf extracts of Passiflora incarnata, P. caerulea and P. alata and in vitro cytotoxic potential on leukemia cell lines

ABSTRACT Passiflora caerulea L., P. alata Curtis and P. incarnata L. (synonym for P. edulis Sims), are the most popular representatives of the Passiflora genus in South America. In recent years, a growing attention is paid to the biological activity and phytochemical profiles of crude extracts from various species of Passiflora in worldwide. The aim of this study was to evaluate and to compare of anti-leukemic activity of the dry crude extracts from leaves of three Passiflora species from greenhouse of Poland in two human acute lymphoblastic leukemia cell lines: CCRF-CEM and its multidrug resistant variant. Two systems of liquid chromatography in order to assessment of phytochemical composition of extracts were applied. Extracts of P. alata and P. incarnata showed the potent inhibitory activity against human acute lymphoblastic leukemia CCRF-CEM, while P. caerulea not showed activity (or activity was poor). Despite similarities in quality phytochemical profile of extracts from P. caerulea and P. incarnata, differences in quantity of chemical compounds may determine their various pharmacological potency. For the activity of P. alata extract the highest content of terpenoids and a lack of flavones C-glycosides are believed to be crucial. Summarizing, the crude extract from P. alata leaves may be considered as a substance for complementary therapy for cancer patients.

Impact of PKCε downregulation on autophagy in glioblastoma cells.

BACKGROUND: Several 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: Effects 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.

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.

Semisynthetic oleanane triterpenoids inhibit migration and invasion of human breast cancer cells through downregulated expression of the ITGB1/PTK2/PXN pathway.

This paper reports a study on the role of two synthetic derivatives of oleanolic acid (OA), HIMOXOL and Br-HIMOLID, in the regulation of cell migration and invasion and the underlying molecular mechanisms of breast cancer cells. The effect of the compounds on four breast cancer cell lines (MCF7, MDA-MB-231, MDA-MB-468, and T-47D) and also on noncancerous breast cells, MCF-12A, was reported. The compounds had no effect on the migration of MCF-12A cells. However, both the derivatives revealed a higher cytotoxicity than the maternal compound OA, and in sub-cytotoxic concentrations, they decreased the migration of MCF7, MDA-MB-231, and MDA-MB-468 breast cancer cells and also the invasion of MCF7 and MDA-MB-231 cells; although, the derivatives had no effect on the migration and invasion of T-47D cells. Both the derivatives of OA inhibited the cell migratory and invasive abilities of breast cancer cells by downregulating the expressions of ITGB1, PTK2, and PXN genes and by decreasing the phosphorylation status and the level of its respective proteins (integrin ß1, FAK, and paxillin, respectively). This study is the first to report the antimigratory and anti-invasive activities of HIMOXOL and Br-HIMOLID in breast cancer cells.

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

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, Ala(159) → 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.

[Autophagy, new perspectives in anticancer therapy]. / Autofagia, nowe perspektywy w terapii przeciwnowotworowej.

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.

Methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate (HIMOXOL), a synthetic oleanolic acid derivative, induces both apoptosis and autophagy in MDA-MB-231 breast cancer cells.

HIMOXOL (methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate) is a synthetic derivative of oleanolic acid (OA). HIMOXOL revealed the highest cytotoxic effect among tested synthetic OA analogs. In this study we focused on elucidating the cytotoxic mechanism of HIMOXOL in MDA-MB-231 breast cancer cells. HIMOXOL reduced MDA-MB-231 cell viability with an IC50 value of 21.08±0.24µM. In contrast to OA, the tested compound induced cell death by activating apoptosis and the autophagy pathways. More specifically, we found that HIMOXOL was able to activate the extrinsic apoptotic pathway, which was proven by observation of caspase-8, caspase-3 and PARP-1 protein activation in Western blot analysis. An increase in the ratio of Bax/Bcl-2 protein levels was also detected. Moreover, HIMOXOL triggered microtubule-associated protein LC3-II expression and upregulated beclin 1. This observed compound activity was modulated by mitogen-activated protein kinases and NFκB/p53 signaling pathways. Together, these data suggest that HIMOXOL, a synthetic oleanolic acid derivative which activates dual cell death machineries, could be a potential and novel chemotherapeutic agent.

Telomerase downregulation induces proapoptotic genes expression and initializes breast cancer cells apoptosis followed by DNA fragmentation in a cell type dependent manner.

The aim of the study was to analyze the consequence of silencing genes coding for the key subunits of the telomerase complex, i.e. TERT, TERC and TP1 in human breast cancer MCF7 and MDA-MB-231cells. The transfection was performed using Lipofectamine2000 and pooled siRNAs. The cytotoxic and/or antiproliferative effect of siRNA was measured by the SRB assay, the cell cycle was analysed by flow cytometry and DNA fragmentation by TUNEL analysis. Telomerase activity was assessed by TRAP, followed by PAGE and ELISA assays. Telomerase downregulation was also assessed using qPCR in order to estimate the changes in the expression profile of genes engaged in apoptosis. It was revealed that treatment of breast cancer cells with different siRNAs (100 nM) resulted in a cell type and time-dependent effects. The downregulation of telomerase subunits was followed by reduction of telomerase activity down to almost 60% compared to control cells. However, a significant effect was only observed when the TERT subunit was downregulated. Its silencing resulted in a significant (p<0.05) increase of apoptosis (over 10% in MCF7 and about 5% in MDA-MB-231 cells, corresponding to the Annexin V assay) and DNA fragmentation (almost 30% in MCF7 and over 25% in MDA-MB-231 cells). Interestingly, also several proapoptotic genes were induced after the downregulation of the key telomerase subunit, including Bax, Bik or caspase-1 and caspase-14, as well as NGFR and TNFSF10 which were upregulated twice and more.

Murine double minute clone 2,309T/G and 285G/C promoter single nucleotide polymorphism as a risk factor for breast cancer: a Polish experience.

BACKGROUND: Breast cancer is a multifactorial disease caused by complex interactions between genetic and environmental factors. Recently, a functional polymorphism, MDM2 285G>C (rs117039649), has been discovered. This polymorphism antagonizes the effect of the 309T>G (rs2279744) polymorphism on the same gene, resulting in decreased MDM2 transcription. METHODS: The MDM2 285G>C and 309T>G polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing analysis in women with breast cancer (n=468) and controls (n=550). RESULTS: The odds ratio (OR) for breast cancer patients with the MDM2 285C/C and 285G/C genotypes was 0.4768 (95% confidence interval [CI] 0.2906-0.7824; p=0.0033, pcorr=0.0066). We also found a significantly lower frequency of the MDM2 285C allele in patients with breast cancer than in controls: the OR for the C allele in patients with breast cancer was 0.4930 (95% CI=0.3059-0.7947, p=0.0031, pcorr=0.0062). The p value of the chi-square test for the trend observed for the MDM2 285G>C polymorphism was statistically significant (ptrend=0.0036). The statistical power of this study amounted to 85% for the G/C or C/C genotypes and 85% for the C allele. However, we did not observe significant differences between the distribution of MDM2 309T>G genotypes and alleles in patients with breast cancer and healthy controls. CONCLUSION: In a sample of the Polish population, we observed that the MDM2 285C gene variant may be a significant protective factor against breast cancer.

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 IC(50) was found to be 17.9 ± 1.6 µM. In HeLa cells overexpressing doxycycline-inducible constitutively active PKCε (HeLaPKCεA/E), the IC(50) 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 24h, 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 24h 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.

Study of ABCB1 polymorphism frequency in breast cancer patients from Poland.

BACKGROUND: The accumulation of mutagenic substances in the human body may result in DNA metabolism disruption followed by carcinogenesis. As a consequence of mutations in the genes coding for transmembrane protein pumps, the intracellular concentration of xenobiotics may significantly increase. This, in turn, may provoke altered risk for cancer development. The gene known to be the most relevant in the transport of numerous compounds is ABCB1 (also known as MDR1). Numerous mutations and polymorphisms that affect the encoded protein's (PgP) function were identified in this gene. The aim of the study was to define the frequency of 2677G>A,T and 3435C>T polymorphisms in a population of Polish breast cancer patients and to estimate their contribution to cancer development. METHODS: The polymorphism frequency analysis (209 patients vs. 202 control subjects) was performed either by allele-specific amplification (2677G>A,T) or by restriction fragment length polymorphism (RFLP) using the SAU3AI restriction enzyme (3435C>T) followed by verification with hybridization probe assays in a Real-Time system and sequencing. RESULTS: In the control group the frequency of individual 2677 genotypes was: wild homozygous GG = 34%, heterozygous G/T or G/A = 52.5% and variant homozygous AA or TT = 13.5%, while the genotype frequency in the group of studied patients was 43.5, 44.5 and 12%, respectively. In the control group, the frequency of individual 3435 genotypes was: CC = 25.4%, CT = 50.2%, TT = 24.4%, while the genotype frequency in the group of studied patients was 23, 46 and 31%, respectively. CONCLUSION: Thus, no significant differences in the studied polymorphism frequencies were observed. It is then suggested that the studied polymorphisms, although probably good candidates in other tissue cancer types, might not be good predictive factors in breast cancer risk or development in Caucasians.

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.

CCL2 -2518 A/G single nucleotide polymorphism as a risk factor for breast cancer.

The contribution of the CCL2 -2518 A>G (rs 1024611) polymorphism in the occurrence and progression of various cancers has been found to be discordant. We studied the prevalence of the CCL2 -2518 A>G polymorphism in patients with breast cancer (n = 160) and controls (n = 323) in a sample of the Polish population. There were no significant differences in CCL2 -2518 A>G genotypes between patients with breast tumors and controls. Odds ratio (OR) for patients bearing the GG genotype was 1.481 (95% CI = 0.7711-2.845, P = 0.2358), and OR of the GG and AG genotypes was 0.7269 (95% CI = 0.4967-1.064, P = 0.1002). There was also no significant distinction in the prevalence of alleles between patients and healthy individuals. OR for the CCL2 -2518 G allele frequency was 0.8903 (95% CI = 0.6611-1.199, P = 0.4441). Analysis of the association between tumor size, lymph node metastases, histological grade, and distribution of genotypes and alleles for the CCL2 -2518 A>G polymorphism also did not show significant differences. Our results did not show association of the CCL2 -2518 A>G polymorphism with breast cancer occurrence and clinical characteristics in a sample of the Polish cohort.

Oleanolic acid derivative methyl 3,11-dioxoolean-12-en-28-olate targets multidrug resistance related to ABCB1.

Multidrug resistance (MDR) in leukemia patients is a great incentive to the development of new drugs. In a search for potential multidrug resistance modulators we tested a group of oleanolic acid (OA) analogues modified at C-3, C-11, C-12 and C-28 using an experimental model consisting of three human acute lymphoblastic leukemia cell lines (CCRF-CEM and the multidrug resistant sublines CCRF-VCR1000 and CCRF-ADR5000). The most effective compound, methyl 3,11-dioxoolean-12-en-28-olate (DIOXOL) was more potent in cell viability inhibition than its precursor - OA, and showed similar or even higher activity in the drug resistant than in the wild-type cells. Resistance factor (RF) values obtained for CCRF-VCR1000 and CCRF-ADR-5000 cells using MTT assay were 0.7 and 0.8 (24 h of treatment) and after 72 h of treatment 0.9 and 1.1, respectively. Moreover, 5 µM DIOXOL significantly reduced the expression of the ABCB1 gene in MDR cells by around 30%, and also decreased the level of P-gp protein. Compared to untreated control cells, DIOXOL treatment resulted in a significant P-gp decrease (30% in CCRF-ADR5000 and 50% in CCRF-VCR1000), that was detected by western blot and confirmed by flow cytometry analysis. Moreover, DIOXOL (at 10 µM) significantly inhibited P-gp transport function by more than twofold comparing to control, untreated cells that was demonstrated using rhodamine 123-based functional test. The compound exhibited synergistic activity with ABCB1 substrate - adriamycin in CCRF-VCR1000 cells, indicating partial but significant MDR reversing ability.

Phytosterols in physiological concentrations target multidrug resistant cancer cells.

Phytosterols have been proposed to act as potent anticancer agents. However the mechanism of their action has not been elucidated yet. Thus, the aim of our study was to determine whether plant sterols and their thermal processing products (in physiological concentration range) could influence the viability of cancer cells and thus could be considered as positive diet complements. Additionally we decided to study potential specificity of those natural compounds against cells showing high multidrug resistance. In this study we show that the cytotoxic effect of ß-sitosterol was observed in both, estrogen-dependent and estrogen-independent cells. It was also shown that the ß-sitosterol was significantly more cytotoxic in cells with basal ABCB1 expression (MCF7) than in multidrug resistant NCI/ADR-RES. Surprisingly, 5a,6a-epoxysitosterol did not decrease the viability of any investigated cells but on the contrary, it provoked their increased proliferation. It was shown that oxyphytosterols blocked the cell cycle of MCF7 cells in G0/G1 phase while did not affect NCI/ADR-RES cell cycle in physiological concentration range. We also show that PgP activity (responsible for Multidrug Resistance phenomena) is inhibited by ß-sitosterol. Thus, the phytosterols are supposed to act at various mechanisms but, what is most interesting, can target cells showing high multidrug resistance potential.

CXCL12-3' G801A polymorphism is not a risk factor for breast cancer.

The CXCL12-3' G801A transition (rs1801157) has been associated with the incidence of breast cancer. However, the contribution of CXCL12-3' G801A polymorphisms in breast cancer development and progression has been controversial. Therefore, we examined the incidence of CXCL12-3' G801A polymorphic variants in patients with breast cancer (n = 193) and controls (n = 199) in a Polish cohort. We observed a trend of slightly increased presence of CXCL12-3' AA and GA genotypes and CXCL12-3'A allele frequency in patients with breast cancer compared with healthy individuals. However, these differences between cases and controls were not statistically significant. Odds ratio (OR) for patients with breast cancer and the CXCL12-3' A/A genotype was 1.898 (95% confidence interval [CI] = 0.6242-5.770, p = 0.2866) and OR of the CXCL12-3' A/A and A/G genotypes was 1.229 (95% CI = 0.8082-1.868, p = 0.3395). OR for the CXCL12-3'A allele frequency was 1.249 (95% CI = 0.8716-1.789, p = 0.2352). Our investigation did not support the CXCL12-3'A gene variant as a risk factor for breast cancer incidence in a sample of the Polish population.

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.