Pygenic Acid A (PA) Sensitizes Metastatic Breast Cancer Cells to Anoikis and Inhibits Metastasis In Vivo.

Metastasis is the main cause of cancer-related deaths. Anoikis is a type of apoptosis caused by cell detachment, and cancer cells become anoikis resistant such that they survive during circulation and can successfully metastasize. Therefore, sensitization of cancer cells to anoikis could prevent metastasis. Here, by screening for anoikis sensitizer using natural compounds, we found that pygenic acid A (PA), a natural compound from Prunella vulgaris, not only induced apoptosis but also sensitized the metastatic triple-negative breast cancer cell lines, MDA-MB-231 cells (human) and 4T1 cells (mouse), to anoikis. Apoptosis protein array and immunoblotting analysis revealed that PA downregulated the pro-survival proteins, including cIAP1, cIAP2, and survivin, leading to cell death of both attached and suspended cells. Interestingly, PA decreased the levels of proteins associated with anoikis resistance, including p21, cyclin D1, p-STAT3, and HO-1. Ectopic expression of active STAT3 attenuated PA-induced anoikis sensitivity. Although PA activated ER stress and autophagy, as determined by increases in the levels of characteristic markers, such as IRE1α, p-elF2α, LC3B I, and LC3B II, PA treatment resulted in p62 accumulation, which could be due to PA-induced defects in autophagy flux. PA also decreased metastatic characteristics, such as cell invasion, migration, wound closure, and 3D growth. Finally, lung metastasis of luciferase-labeled 4T1 cells decreased following PA treatment in a syngeneic mouse model when compared with the control. These data suggest that PA sensitizes metastatic breast cancer cells to anoikis via multiple pathways, such as inhibition of pro-survival pathways and activation of ER stress and autophagy, leading to the inhibition of metastasis. These findings suggest that sensitization to anoikis by PA could be used as a new therapeutic strategy to control the metastasis of breast cancer.

Proteome-wide identification of arginine methylation in colorectal cancer tissues from patients.

BACKGROUND: Protein arginine methylation reaction is catalyzed by protein arginine methyltransferase (PRMT) and the modification is implicated in various diseases including cancer. Currently, thousands of arginine methylation sites have been identified using high-resolution mass spectrometry-based proteomics technology. However, identification of arginine methylation using clinical samples at proteome level has not been reported yet. The objective of the present study was to identify, monomethyl-arginine (MMA) and asymmetric dimethyl-arginine (ADMA) sites in colorectal cancer (CRC) tissues at proteome level. METHODS: Pooled CRC tissue samples from 10 patients with stage II and III were digested by trypsin and these digests were further processed and lyophilized. Using monomethyl- or asymmetric dimethyl arginine (MMA or ADMA, respectively) motif kits, methylarginine-containing peptides were enriched and subsequently analyzed by high-resolution LC-MS/MS. DLD1 and HCT116 colon cancer cells were treated with type I PRMTs inhibitor (MS023) alone or combined with SN-38, and the effect of the drugs on CRC cell proliferation and apoptosis was measured by water-soluble tetrazolium salt (WST-1) assay and FACS analysis, respectively. RESULTS: In the present study, 455 MMA sites of 272 proteins and 314 ADMA sites of 155 proteins were identified from CRC tissues acquired from patients. In addition, 216 methylation sites and 75 substrates for PRMTs were newly identified. These results reveal the significant presence of MMA and ADMA sites on nucleic acid binding proteins and protein complexes involved in transcription. To investigate the effect of protein arginine methylation in CRC proliferation and apoptosis, MS023 was treated to two CRC cell lines. After 48 h treatment with various concentrations of MS023, CRC cell proliferation was significantly suppressed, with concomitant apoptosis induction. Furthermore, MS023 treatment significantly enhanced the inhibitory effect of SN-38 on CRC cell proliferation. CONCLUSION: This work reports the first comprehensive analysis of arginine methylation with clinical sample and suggests that type I PRMTs are potential therapeutic targets for drug discovery in CRC.

The prognostic significance of protein arginine methyltransferase 6 expression in colon cancer.

Protein arginine methylation is involved in cellular differentiation and proliferation. Recently, aberrant expression of protein arginine methyltransferases, which are responsible for the methylation reaction, has been reported in various types of cancer. However, there is no clear evidence regarding the prognostic value of abnormal PRMT6 expression in colorectal cancer or the effect of PRMT6 regulation on CRC cells. We investigated the expression patterns of PRMT6 in patients with stage II and III CRC. We detected nuclear expression of PRMT6 in 23.7% of carcinoma samples by immunohistochemistry. Among the clinicopathological parameters, the ratio of poorly differentiated cancer cells was approximately two-fold higher in patients with PRMT6-positive disease than in those with PRMT6-negative disease (p = 0.002). Patients with PRMT6-positive CRC had a shorter disease-free survival than those with PRMT6-negative CRC in both univariate and multivariate analyses (p = 0.018 and p = 0.035, respectively). siRNA-mediated inhibition of PRMT6 expression in CRC cells induced p21WAF1/CIP1 overexpression and suppressed cell growth and colony-forming ability. Concomitantly, apoptosis was induced in PRMT6-suppressed CRC cells. These data suggest that PRMT6 can serve as a biomarker for unfavorable prognosis and as a therapeutic target in CRC.

Prenatal hyperbaric normoxia treatment improves healthspan and regulates chitin metabolic genes in Drosophila melanogaster.

Aging is a universal, irreversible process accompanied by physiological declines that culminate in death. Rapid progress in gerontology research has revealed that aging can be slowed through mild stress-induced hormesis. We previously reported that hyperbaric normoxia (HN, 2 atm absolute pressure with 10% O2) induces a cytoprotective response in vitro by regulating fibronectin. In the present study, we investigated the hormetic effects of prenatal HN exposure on Drosophila healthspan related to molecular defense mechanisms. HN exposure had no disruptive effect on developmental rate or adult body weight. However, lifespan was clearly enhanced, as was resistance to oxidative and heat stress. In addition, levels of reactive oxygen species were significantly decreased and motor performance was increased. HN stress has been shown to trigger molecular changes in the heat shock response and ROS scavenging system, including hsp70, catalase, glutathione synthase, and MnSOD. Furthermore, to determine the hormetic mechanism underlying these phenotypic and molecular changes, we performed a genome-wide profiling in HN-exposed and control flies. Genes encoding chitin metabolism were highly up-regulated, which could possibly serve to scavenge free radicals. These results identify prenatal HN exposure as a potential hormetic factor that may improve longevity and healthspan by enhancing defense mechanisms in Drosophila.

Nmdmc overexpression extends Drosophila lifespan and reduces levels of mitochondrial reactive oxygen species.

NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is a bifunctional enzyme involved in folate-dependent metabolism and highly expressed in rapidly proliferating cells. However, Nmdmc physiological roles remain unveiled. We found that ubiquitous Nmdmc overexpression enhanced Drosophila lifespan and stress resistance. Interestingly, Nmdmc overexpression in the fat body was sufficient to increase lifespan and tolerance against oxidative stress. In addition, these conditions coincided with significant decreases in the levels of mitochondrial ROS and Hsp22 as well as with a significant increase in the copy number of mitochondrial DNA. These results suggest that Nmdmc overexpression should be beneficial for mitochondrial homeostasis and increasing lifespan.