Background: Rac is a central regulator of cancer cell migration/invasion and metastasis. Results: EHop-016 inhibits Rac activity with an IC 50 of 1 M. EHop-016 blocks Rac interaction with the Rac exchange factor Vav2, lamellipodia extension, and cell migration. Conclusion: EHop-016 is an effective Rac inhibitor. Significance: EHop-016 has potential as a metastasis therapeutic and for investigations of Rac-regulated cellular responses.
Alteration in cytoskeletal organization appears to underlie mechanisms of gravity sensitivity in space-flown cells. Human T lymphoblastoid cells (Jurkat) were flown on the Space Shuttle to test the hypothesis that growth responsiveness is associated with microtubule anomalies and mediated by apoptosis. Cell growth was stimulated in microgravity by increasing serum concentration. After 4 and 48 h, cells filtered from medium were fixed with formalin. Post-flight, confocal microscopy revealed diffuse, shortened microtubules extending from poorly defined microtubule organizing centers (MTOCs). In comparable ground controls, discrete microtubule filaments radiated from organized MTOCs and branched toward the cell membrane. At 4 h, 30% of flown, compared to 17% of ground, cells showed DNA condensation characteristic of apoptosis. Time-dependent increase of the apoptosis-associated Fas/ APO-1 protein in static flown, but not the in-flight 1 g centrifuged or ground controls, confirmed microgravity-associated apoptosis. By 48 h, ground cultures had increased by 40%. Flown populations did not increase, though some cells were cycling and actively metabolizing glucose. We conclude that cytoskeletal alteration, growth retardation, and metabolic changes in space-flown lymphocytes are concomitant with increased apoptosis and time-dependent elevation of Fas/APO-1 protein. We suggest that reduced growth response in lymphocytes during spaceflight is linked to apoptosis.
Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 microM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 microM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.
The Rho GTPases Rac (Ras-related C3 botulinum toxin substrate) and Cdc42 (cell division control protein 42 homolog) regulate cell functions governing cancer malignancy, including cell polarity, migration, and cell cycle progression. Accordingly, our recently developed Rac inhibitor EHop-016 (IC50, 1,100 nM) inhibits cancer cell migration and viability, and reduces tumor growth, metastasis, and angiogenesis in vivo. Herein, we describe MBQ-167, which inhibits Rac and Cdc42 with IC50s of 103 nM and 78 nM respectively, in metastatic breast cancer cells. Consequently, MBQ-167 significantly decreases Rac and Cdc42 downstream effector p21-activated kinase (PAK) signaling and the activity of signal transducer and activator of transcription (STAT3), without affecting Rho, MAPK, or Akt activities. MBQ-167 also inhibits breast cancer cell migration, viability, and mammosphere formation. Moreover, MBQ-167 affects cancer cells that have undergone epithelial to mesenchymal transition by a loss of cell polarity, and inhibition of cell surface actin-based extensions, to ultimately result in detachment from the substratum. Prolonged incubation (120 h) in MBQ-167 decreases metastatic cancer cell viability with a GI50 of ~130 nM, without affecting non-cancer mammary epithelial cells. The loss in cancer cell viability is due to MBQ-167-mediated G2/M cell cycle arrest and subsequent apoptosis, especially of the detached cells. In vivo, MBQ-167 inhibits mammary tumor growth and metastasis in immunocompromised mice by ~90%. In conclusion, MBQ-167 is 10X more potent than other currently available Rac/Cdc42 inhibitors, and has potential to be developed as an anticancer drug, as well as a dual inhibitory probe for the study of Rac and Cdc42.
The cancer preventive properties of grape products such as red wine have been attributed to polyphenols enriched in red wine. However, much of the studies on cancer preventive mechanisms of grape polyphenols have been conducted with individual compounds at concentrations too high to be achieved via dietary consumption. We recently reported that combined grape polyphenols at physiologically relevant concentrations are more effective than individual compounds at inhibition of ERα(−), ERβ(+) MDA-MB-231 breast cancer cell proliferation, cell cycle progression, and primary mammary tumor growth (Schlachterman et al., Transl Oncol 1:19–27, 2008). Herein, we show that combined grape polyphenols induce apoptosis and are more effective than individual resveratrol, quercetin, or catechin at inhibition of cell proliferation, cell cycle progression, and cell migration in the highly metastatic ER (−) MDA-MB-435 cell line. The combined effect of dietary grape polyphenols (5 mg/kg each resveratrol, quercetin, and catechin) was tested on progression of mammary tumors in nude mice created from green fluorescent protein-tagged MDA-MB-435 bone metastatic variant. Fluorescence image analysis of primary tumor growth demonstrated a statistically significant decrease in tumor area by dietary grape polyphenols. Molecular analysis of excised tumors demonstrated that reduced mammary tumor growth may be due to upregulation of FOXO1 (forkhead box O1) and NFKBIA (IκBα), thus activating apoptosis and potentially inhibiting NfκB (nuclear factor κB) activity. Image analysis of distant organs for metastases demonstrated that grape polyphenols reduced metastasis especially to liver and bone. Overall, these results indicate that combined dietary grape polyphenols are effective at inhibition of mammary tumor growth and site-specific metastasis.
Inflammatory breast cancer (IBC) is the most lethal and least understood form of advanced breast cancer. Its lethality originates from its nature of invading the lymphatic system and absence of a palpable tumor mass. Different from other metastatic breast cancer cells, IBC cells invade by forming tumor spheroids that retain E-cadherin-based cell–cell adhesions. Herein we describe the potential of the medicinal mushroom Ganoderma lucidum (Reishi) as an attractive candidate for anti-IBC therapy. Reishi contains biological compounds that are cytotoxic against cancer cells. We report the effects of Reishi on viability, apoptosis, invasion, and its mechanism of action in IBC cells (SUM-149). Results show that Reishi selectively inhibits cancer cell viability although it does not affect the viability of noncancerous mammary epithelial cells. Apoptosis induction is consistent with decreased cell viability. Reishi inhibits cell invasion and disrupts the cell spheroids that are characteristic of the IBC invasive pathology. Reishi decreases the expression of genes involved in cancer cell survival and proliferation (BCL-2, TERT, PDGFB), and invasion and metastasis (MMP-9), whereas it increases the expression of IL8. Reishi reduces BCL-2, BCL-XL, E-cadherin, eIF4G, p120-catenin, and c-Myc protein expression and gelatinase activity. These findings suggest that Reishi is an effective anti-IBC therapeutic.
The medicinal mushroom Ganoderma lucidum (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using in vivo and in vitro IBC models. IBC is a lethal and aggressive form of breast cancer that manifests itself without a typical tumor mass. Studies show that IBC tissue biopsies overexpress E-cadherin and the eukaryotic initiation factor 4GI (eIF4GI), two proteins that are partially responsible for the unique pathological properties of this disease. IBC is treated with a multimodal approach that includes non-targeted systemic chemotherapy, surgery, and radiation. Because of its non-toxic and selective anti-cancer activity, medicinal mushroom extracts have received attention for their use in cancer therapy. Our previous studies demonstrate these selective anti-cancer effects of Reishi, where IBC cell viability and invasion, as well as the expression of key IBC molecules, including eIF4G is compromised. Thus, herein we define the mechanistic effects of Reishi focusing on the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, a regulator of cell survival and growth. The present study demonstrates that Reishi treated IBC SUM-149 cells have reduced expression of mTOR downstream effectors at early treatment times, as we observe reduced eIF4G levels coupled with increased levels of eIF4E bound to 4E-BP, with consequential protein synthesis reduction. Severe combined immunodeficient mice injected with IBC cells treated with Reishi for 13 weeks show reduced tumor growth and weight by ∼50%, and Reishi treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G, and p70S6K, and activity of extracellular regulated kinase (ERK1/2). Our results provide evidence that Reishi suppresses protein synthesis and tumor growth by affecting survival and proliferative signaling pathways that act on translation, suggesting that Reishi is a potential natural therapeutic for breast and other cancers.
Cytoskeletal disruption and growth arrest consistently occur in space‐flown human acute leukemic T cells (Jurkat). Although the microtubules appear to reorganize during spaceflight, cells remain nonproliferative. To test the hypothesis that spaceflight alters cytoskeletal gene expression and may thus affect cytoskeletal function, we flew Jurkat cells on Space Transportation System (STS) 95 and compared RNA message by cDNA microarray in space‐flown vs. ground controls at 24 h (4,324 genes) and 48 h (>20,000 genes). Messages for 11 cytoskeleton‐related genes, including calponin, dynactin, tropomodulin, keratin 8, two myosins, an ankyrin EST, an actinlike protein, the cytoskeletal linker (plectin), and a centriole‐associated protein (C‐NAP1), were up‐regulated in space‐flown compared with ground control cells; gelsolin precursor was down‐regulated. Up‐regulation of plectin and C‐NAP1 message in both space‐flown cells and vibrated controls is a novel finding and implies their role in vibration damage repair. This first report of cDNA microarray screening of gene expression in space‐flown leukemic T cells also identifies differential expression of genes that regulate growth, metabolism, signal transduction, adhesion, transcription, apoptosis, and tumor suppression. Based on differential expression of cytoskeletal genes, we conclude that centriole‐centriole, membrane‐cytoskeletal, and cytoskeletal filament associations are altered in the orbital phase of spaceflight.
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