Anti-Metastatic and Anti-Tumor Growth Effects of Origanum majorana on Highly Metastatic Human Breast Cancer Cells: Inhibition of NFκB Signaling and Reduction of Nitric Oxide Production

Background

We have recently reported that Origanum majorana exhibits anticancer activity by promoting cell cycle arrest and apoptosis of the metastatic MDA-MB-231 breast cancer cell line. Here, we extended our study by investigating the effect of O . majorana on the migration, invasion and tumor growth of these cells.

Results

We demonstrate that non-cytotoxic concentrations of O . majorana significantly inhibited the migration and invasion of the MDA-MB-231 cells as shown by wound-healing and matrigel invasion assays. We also show that O . majorana induce homotypic aggregation of MDA-MB-231 associated with an upregulation of E-cadherin protein and promoter activity. Furthermore, we show that O . majorana decrease the adhesion of MDA-MB-231 to HUVECs and inhibits transendothelial migration of MDA-MB-231 through TNF-α-activated HUVECs. Gelatin zymography assay shows that O . majorana suppresses the activities of matrix metalloproteinase-2 and -9 (MMP-2 and MMP-9). ELISA, RT-PCR and Western blot results revealed that O . majorana decreases the expression of MMP-2, MMP-9, urokinase plasminogen activator receptor (uPAR), ICAM-1 and VEGF. Further investigation revealed that O . majorana suppresses the phosphorylation of IκB, downregulates the nuclear level of NFκB and reduces Nitric Oxide (NO) production in MDA-MB-231 cells. Most importantly, by using chick embryo tumor growth assay, we also show that O . majorana promotes inhibition of tumor growth and metastasis in vivo.

Conclusion

Our findings identify Origanum majorana as a promising chemopreventive and therapeutic candidate that modulate breast cancer growth and metastasis.     

Concerted Suppression of STAT3 and GSK3β Is Involved in Growth Inhibition of Non-Small Cell Lung Cancer by Xanthatin

Xanthatin, a sesquiterpene lactone purified from Xanthium strumarium L., possesses prominent anticancer activity. We found that disruption of GSK3β activity was essential for xanthatin to exert its anticancer properties in non-small cell lung cancer (NSCLC), concurrent with preferable suppression of constitutive activation of STAT3. Interestingly, inactivation of the two signals are two mutually exclusive events in xanthatin-induced cell death. Moreover, we surprisingly found that exposure of xanthatin failed to trigger the presumable side effect of canonical Wnt/β-Catenin followed by GSK3β inactivation. We further observed that the downregulation of STAT3 was required for xanthatin to fine-tune the risk. Thus, the discovery of xanthatin, which has ability to simultaneously orchestrate two independent signaling cascades, may have important implications for screening promising drugs in cancer therapies.     

JAK/STAT3-Regulated Fatty Acid β-Oxidation Is Critical for Breast Cancer Stem Cell Self-Renewal and Chemoresistance

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Evaluation of STAT3 Signaling in ALDH+ and ALDH+/CD44+/CD24? Subpopulations of Breast Cancer Cells

Background

STAT3 activation is frequently detected in breast cancer and this pathway has emerged as an attractive molecular target for cancer treatment. Recent experimental evidence suggests ALDH-positive (ALDH⁺), or cell surface molecule CD44-positive (CD44⁺) but CD24-negative (CD24⁻) breast cancer cells have cancer stem cell properties. However, the role of STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells is unknown.

Methods and Results

We examined STAT3 activation in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells by sorting with flow cytometer. We observed ALDH-positive (ALDH⁺) cells expressed higher levels of phosphorylated STAT3 compared to ALDH-negative (ALDH⁻) cells. There was a significant correlation between the nuclear staining of phosphorylated STAT3 and the expression of ALDH1 in breast cancer tissues. These results suggest that STAT3 is activated in ALDH⁺ subpopulations of breast cancer cells. STAT3 inhibitors Stattic and LLL12 inhibited STAT3 phosphorylation, reduced the ALDH⁺ subpopulation, inhibited breast cancer stem-like cell viability, and retarded tumorisphere-forming capacity in vitro. Similar inhibition of STAT3 phosphorylation, and breast cancer stem cell viability were observed using STAT3 ShRNA. In addition, LLL12 inhibited STAT3 downstream target gene expression and induced apoptosis in ALDH⁺ subpopulations of breast cancer cells. Furthermore, LLL12 inhibited STAT3 phosphorylation and tumor cell proliferation, induced apoptosis, and suppressed tumor growth in xenograft and mammary fat pad mouse models from ALDH⁺ breast cancer cells. Similar in vitro and tumor growth in vivo results were obtained when ALDH⁺ cells were further selected for the stem cell markers CD44⁺ and CD24⁻.

Conclusion

These studies demonstrate an important role for STAT3 signaling in ALDH⁺ and ALDH⁺/CD44⁺/CD24⁻ subpopulations of breast cancer cells which may have cancer stem cell properties and suggest that pharmacologic inhibition of STAT3 represents an effective strategy to selectively target the cancer stem cell-like subpopulation.     

Suppressive Effects of Genistein on Oxidative Stress and NFκB Activation in RAW 264.7 Macrophages

This study was designed to investigate whether genistein may ameliorate oxidative stress and nuclear factor κB (NFκB) activation in the lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophage cell line. Treatment of RAW 264.7 cells with genistein significantly reduced lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in a dose-dependent manner with an IC₅₀ of 69.4 μM. Genistein at 50 μM and 100 μM concentrations reduced thiobarbituric acid-reactive substances (TBARS) accumulation, increasing the GSH level and antioxidant enzyme activities, such as superoxide dismutase (SOD) and catalase. The specific DNA-binding activities of nuclear factor κB (NFκB) on nuclear extracts from 50 μM and 100 μM genistein treatments were significanly suppressed. These results suggest that genistein has mild antioxidant activity to suppress intracellular oxidative stress and NFκB activation.     

Effects of 3-Methylgibberellin Analogs on Gibberellin 3β-Hydroxylases and Plant Growth

Six 3-methylgibberellin analogs were synthesized, and their effects on the GA 3β-hydroxylases from immature seeds of Phaseolus vulgaris and Cucurbita maxima, and/or on the growth of dwarf rice (Oryza sativa L. cv. Tan-ginbozu) and cucumber (Cucumis sativus L. cv. Spacemaster) were investigated. 3-Methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉· inhibited the conversion of [2, 3-³H2]GA₉ to [2-³H]GA₄ by GA 3β-hydroxylases from both P. vulgaris and C. maxima at 3 μM and higher. Their C/D-ring-rearranged isomers, 2, 3-didehydro-3-methyl-DGC and 16-deoxo-2, 3-didehydro-3-methyl-DGC, inhibited 3β-hydroxylation by the enzyme from P. vulgaris threefold more strongly than the non-C/D-ring-rearranged compounds, but exhibited no effect on 3β-hydroxylation by the enzyme from C. maxima. In a dwarf rice seedling assay, 3-methyl-GA₅ and 2, 3-didehydro-3-methyl-GA₉ promoted shoot elongation at doses of 300 ng/plant and higher, and 3α-methyl-GA₁ and 3α-methyl-GA₄ at doses of 30 ng/plant and higher. In contrast 2, 3-didehydro-3-methyl-DGC inhibited shoot growth to half that of the control at a dose of 300 ng/plant, and 16-deoxo-2, 3-didehydro-3-methyl-DGC showed no effect on growth. In a cucumber seedling assay, 3α-methyl-GA₄ promoted hypocotyl elongation at doses of 300 ng/plant and higher. The other C-3 methyl compounds showed no effect on the hypocotyl elongation of cucumber seedlings.     

Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.     

Educational Differences in Postmenopausal Breast Cancer – Quantifying Indirect Effects through Health Behaviors,Body Mass Index and Reproductive Patterns

Studying mechanisms underlying social inequality in postmenopausal breast cancer is important in order to develop prevention strategies. Standard methods for investigating indirect effects, by comparing crude models to adjusted, are often biased. We applied a new method enabling the decomposition of the effect of educational level on breast cancer incidence into indirect effects through reproductive patterns (parity and age at first birth), body mass index and health behavior (alcohol consumption, physical inactivity, and hormone therapy use). The study was based on a pooled cohort of 6 studies from the Copenhagen area including 33,562 women (1,733 breast cancer cases) aged 50–70 years at baseline. The crude absolute rate of breast cancer was 399 cases per 100,000 person-years. A high educational level compared to low was associated with 74 (95% CI 22–125) extra breast cancer cases per 100,000 person-years at risk. Of these, 26% (95% CI 14%–69%) could be attributed to alcohol consumption. Similar effects were observed for age at first birth (32%; 95% CI 10%–257%), parity (19%; 95%CI 10%–45%), and hormone therapy use (10%; 95% CI 6%–18%). Educational level modified the effect of physical activity on breast cancer. In conclusion, this analysis suggests that a substantial number of the excess postmenopausal breast cancer events among women with a high educational level compared to a low can be attributed to differences in alcohol consumption, use of hormone therapy, and reproductive patterns. Women of high educational level may be more vulnerable to physical inactivity compared to women of low educational level.     

Breast Cancer “Tailored Follow-up” in Italian Oncology Units: A Web-Based Survey

Purpose

Breast cancer follow-up procedures after primary treatment are still a controversial issue. Aim of this study was to investigate, through a web-based survey, surveillance methodologies selected by Italian oncologists in everyday clinical practice.

Methods

Referents of Italian medical oncology units were invited to participate to the study via e-mail through the SurveyMonkey website. Participants were asked how, in their institution, exams of disease staging and follow-up are planned in asymptomatic women and if surveillance continues beyond the 5^th year.

Results

Between February and May 2013, 125 out of 233 (53.6%) invited referents of Italian medical oncology units agreed to participate in the survey. Ninety-seven (77.6%) referents state that modalities of breast cancer follow-up are planned according to the risk of disease progression at diagnosis and only 12 (9.6%) oncology units apply the minimal follow-up procedures according to international guidelines. Minimal follow-up is never applied in high risk asymptomatic women. Ninety-eight (78.4%) oncology units continue follow-up in all patients beyond 5 years.

Conclusions

Our survey shows that 90.4% of participating Italian oncology units declare they do not apply the minimal breast cancer follow-up procedures after primary treatment in asymptomatic women, as suggested by national and international guidelines. Interestingly, about 80.0% of interviewed referents performs the so called “tailored follow-up”, high intensity for high risk, low intensity for low risk patients. There is an urgent need of randomized clinical trials able to determine the effectiveness of risk-based follow-up modalities, their ideal frequency and persistence in time.     

Combinatorial Effects of Nonsteroidal Anti-inflammatory Drugs and Food Constituents on Production of Prostaglandin E2 and Tumor Necrosis Factor-α in RAW264.7 Murine Macrophages

Combinatorial chemopreventive strategies, in contrast to those with individual agents, show potential in terms of potentially lower toxicity and higher efficacy. In this study, we combined several agents and examined their suppressive effects on the combined lipopolysaccharide (LPS)- and interferon(IFN)-γ-induced formation of proinflammatory mediators, including prostaglandin (PG) E₂ and tumor necrosis factor (TNF)-α, in RAW264.7 murine macrophages. The combinatorial effects of indomethacin/genistein (GEN) and aspirin/GEN were found to be synergistic for PGE₂ suppression, while the nimesulide/GEN combination was antagonistic. Further, while (?)-epigallocatechin gallate (EGCG) alone increased LPS/IFM-γ-induced production of PGE₂ and TNF-α as well as cyclooxygenase-2 expression, the EGCG/GEN combination markedly suppressed these parameters. Our results suggest that certain chemopreventive agents act complexly and that, when used in combination, they affect the intracellular signaling pathways of the paired agents to exert additive, synergistic, or antagonistic effects.     

Effects of Experimental Choices and Analysis Noise on Surveys of the “Rare Biosphere”

When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6, and V6-V7 variable regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Pronounced differences between primer sets, however, occurred in the “rare biosphere” involving taxa that we observed in fewer than 11 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest-neighbor approach to directly classifying sequences with a naïve Bayesian algorithm. Again, we observed pronounced differences between these analysis schemes in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere, both the experimental conditions and analysis choices will have a profound impact on the observed results.For nearly 3 decades, investigations of the distribution of microbes in complex environments have focused on the use of rRNA genes (1, 2, 4, 11, 16, 18, 19, 22, 24). Because the full-length 16S rRNA sequence can be obtained with paired-end reads via traditional Sanger sequencing, until recently most studies of the 16S rRNA gene captured most or nearly most of the 16S sequence length. New pyrosequencing technologies, however, have recently been introduced that greatly reduce the per base cost of sequencing but with shorter read lengths than traditional Sanger sequencing (17). This new approach has proven powerful, yielding a previously unobtainable view of rare taxa (7, 12-14, 25).The shorter reads produced by pyrosequencing require the choice of a particular region of the 16S rRNA gene to target for pyrosequencing as well as the choice of an algorithm to classify the taxonomy of the shorter reads. In their initial surveys of microbial diversity with pyrosequencing (12, 14, 25), Sogin and colleagues targeted the V6 variable region, in part because it is was small enough to be captured with the 100-bp reads of the pyrosequencing technology available at the time. Recently, the read length of 454 pyrosequencing machines has been increased to an average of ∼250 bp. This allows for more flexibility in primer design and opens up the possibility of targeting regions of the 16S rRNA gene other than V6. In recent work, Huse et al. took advantage of this new capability to compare the classifications made for the human gut microbiome with the V6 and longer V3 regions (13). Plotting the taxonomic abundance of these two sequence sets against each other yielded an excellent correlation (r² = 0.99), suggesting that the choice of which variable region to target makes little difference. In this report, we introduce a data set examining the performance of sets of primers targeting the V1-V2, V6, and V6-V7 regions. By using a sample for which we have also generated a whole-genome shotgun sequencing run with 250 bp reads, we were able to compare the observed 16S rRNA genes in samples with and without an initial PCR step targeting the 16S rRNA gene. Our results demonstrate that experimental choices such as which region of the 16S rRNA gene to sequence and which algorithm to use to classify taxa are much more likely to affect observations of the “rare biosphere” than more commonly observed taxa.