Metastasis Inhibition.

Cancer metastasis is a common biological phenomenon observed in malignant tumors that can lead to death in affected individuals [...].

Metastasis of Breast Cancer Promoted by Circadian Rhythm Disruption due to Light/Dark Shift and its Prevention by Dietary Quercetin in Mice.

Epidemiological studies have indicated that a disturbed circadian rhythm resulting from night-shift work is a potential risk factor for breast cancer. However, the mechanism of increased risk of breast cancer by night-shift work remains unclear, and there have been few in vivo studies conducted to definitively associate the two factors. In this study, BJMC3879Luc2 mouse breast cancer cells were transplanted into BALB/c mice. Mice were maintained under lighting conditions that modeled the two-shift system and were investigated for the effect of light/dark cycle disruption on tumor growth and lymph node metastasis. Circadian dysfunction, which was confirmed by measuring circadian locomotor activities using a nano tag device in our light/dark shift model, did not affect tumor growth. However, a significant increase in the number of lymph nodes with distant metastasis was observed. Neutrophil-to-lymphocyte ratio, which is an adverse prognostic factor of breast cancer and also indicator of inflammation, also increased. It has been demonstrated that a chronic inflammatory response is associated with cancer malignancy and poor prognosis in various cancers. These results suggest that night-shift work may also affect distant metastasis and prognosis. In addition, we investigated whether dietary quercetin has anti-metastatic activity against light/dark shift-induced metastasis. A diet containing 0.3 % quercetin significantly inhibited distant lymph node metastasis, particularly metastasis to the iliac and kidney lymph nodes. Our results contribute to our understandings of the effects of the external light environment on breast cancer metastasis and provide a glimpse into potential protective effects of dietary quercetin on light/dark disturbance-induced metastasis.

Soluble Vegfr3 gene therapy suppresses multi-organ metastasis in a mouse mammary cancer model.

Accumulating evidence on the association of VEGF-C with lymphangiogenesis and lymph node metastasis implicates lymphatic vessels as a potential target in anti-cancer therapy. To evaluate whether blocking VEGF-C and VEGFR-3 signaling can inhibit multi-organ metastases, a mouse metastatic mammary cancer model was subjected to gene therapy using a soluble VEGFR-3 expression vector (psVEGFR-3). We showed that psVEGFR-3 significantly diminished cell growth in vitro with or without added VEGF-C, and significantly reduced primary tumor growth and tumor metastases to wide-spectrum organs in vivo. Although apoptotic cell death and angiogenesis levels did not differ between the control and psVEGFR-3 groups, cell proliferation and lymphangiogenesis in the mammary tumors were significantly decreased in the psVEGFR-3 group. Furthermore, lymphatic vessel invasion was significantly inhibited in this group. Real-time RT-PCR analysis revealed significantly high expression of the Vegfr3 gene due to gene therapy, and the transcriptional levels of Pcna and Lyve1 tended to decrease in the psVEGFR-3 group. Immunofluorescence staining indicated that phospho-tyrosine expression was considerably lower in tumor cells of psVEGFR-3-treated mammary carcinomas than those of control tumors. Double immunofluorescence staining indicated that phospho-tyrosine+ /LYVE-1+ (a lymphatic vessel marker) tended to decrease in psVEGFR-3-treated mammary carcinomas compared with control mice, indicating a decline in the activity of the VEGF-C/VEGFR-3 axis. These findings showed that a blockade of VEGF-C/VEGFR-3 signaling caused by sVEGFR-3 sequestered VEGF-C and prevented the side-effects of anti-angiogenesis and suppressed overall metastases, suggesting their high clinical significance.

Indomethacin can induce cell death in rat gastric parietal cells through alteration of some apoptosis- and autophagy-associated molecules.

In clinical medicine, indomethacin (IND, a non-steroidal anti-inflammatory drug) is used variously in the treatment of severe osteoarthritis, rheumatoid arthritis, gouty arthritis or ankylosing spondylitis. A common complication found alongside the therapeutic characteristics is gastric mucosal damage. This complication is mediated through apoptosis and autophagy of the gastrointestinal mucosal epithelium. Apoptosis and autophagy are critical homeostatic pathways catalysed by caspases downstream of the gastrointestinal mucosal epithelial injury. Both act through molecular signalling pathways characterized by the initiation, mediation, execution and regulation of the cell regulatory cycle. In this study we hypothesized that dysregulated apoptosis and autophagy are associated with IND-induced gastric damage. We examined the spectra of in vivo experimental gastric ulcers in male Sprague-Dawley rats through gastric gavage of IND. Following an 18-hour fast, IND was administered to experimental rats. They were sacrificed at 3-, 6- and 12-hour intervals. Parietal cells (H+ , K+ -ATPase ß-subunit assay) and apoptosis (TUNEL assay) were determined. The expression of apoptosis-signalling caspase (caspases 3, 8, 9 and 12), DNA damage (anti-phospho-histone H2A.X) and autophagy (MAP-LC3, LAMP-1 and cathepsin B)-related molecules in gastric mucosal cells was examined. The administration of IND was associated with gastric mucosal erosions and ulcerations mainly involving the gastric parietal cells (PCs) of the isthmic and upper neck regions and a time-dependent gradual increase in the number of apoptotic PCs with the induction of both apoptotic (upregulation of caspases 3 and 8) cell death and autophagic (MAP-LC3-II, LAMP-1 and cathepsin B) cell death. Autophagy induced by fasting and IND 3 hours initially prompted the degradation of caspase 8. After 6 and 12 hours, damping down of autophagic activity occurred, resulting in the upregulation of active caspase 8 and its nuclear translocation. In conclusion we report that IND can induce time-dependent apoptotic and autophagic cell death of PCs. Our study provides the first indication of the interactions between these two homeostatic pathways in this context.

Crude α-Mangostin Suppresses the Development of Atherosclerotic Lesions in Apoe-Deficient Mice by a Possible M2 Macrophage-Mediated Mechanism.

Lifestyle choices play a significant role in the etiology of atherosclerosis. Male Apoe-/- mice that develop spontaneous atherosclerotic lesions were fed 0%, 0.3%, and 0.4% mangosteen extracts, composed largely of α-mangostin (MG), for 17 weeks. Body weight gains were significantly decreased in both MG-treated groups compared to the control, but the general condition remained good throughout the study. The levels of total cholesterol (decreased very-low-density lipoprotein in lipoprotein profile) and triglycerides decreased significantly in the MG-treated mice in conjunction with decreased hepatic HMG-CoA synthase and Fatty acid transporter. Additionally, increased serum lipoprotein lipase activity and histopathology further showed a significant reduction in atherosclerotic lesions at both levels of MG exposure. Real-time PCR analysis for macrophage indicators showed a significant elevation in the levels of Cd163, an M2 macrophage marker, in the lesions of mice receiving 0.4% MG. However, the levels of Nos2, associated with M1 macrophages, showed no change. In addition, quantitative immunohistochemical analysis of macrophage subtypes showed a tendency for increased M2 populations (CD68⁺/CD163⁺) in the lesions of mice given 0.4% MG. In further analysis of the cytokine-polarizing macrophage subtypes, the levels of Interleukin13 (Il13), associated with M2 polarization, were significantly elevated in lesions exposed to 0.4% MG. Thus, MG could suppress the development of atherosclerosis in Apoe-/- mice, possibly through an M2 macrophage-mediated mechanism.

Synthetic α-mangostin dilaurate strongly suppresses wide-spectrum organ metastasis in a mouse model of mammary cancer.

We previously reported that, in a mouse model of mammary cancer, α-mangostin alone exhibits anti-metastatic properties. To enhance this anti-metastatic effect, we examined the efficacy of synthetic α-mangostin dilaurate (MGD), prepared by adding lauric acid to α-mangostin, in the same experimental system wherein mice bearing mammary tumors are exposed to dietary MGD at 0, 2000 and 4000 ppm. Lauric acid has a high propensity for lymphatic absorption, which is the most common pathway of initial dissemination of many solid malignancies. Both mammary tumor volumes and wide-spectrum organ metastasis were markedly reduced at 2000 and 4000 ppm: furthermore, survival in the 4000-ppm group was significantly greater than in control mice. Apoptosis in mammary carcinomas was also significantly increased in the 4000-ppm group, whereas blood microvessel density and lymphatic vessel invasion were markedly reduced. In real-time PCR analyses of tumor samples, increased p21 and decreased Pcna expression were observed with 4000 ppm but values were not statistically significant when compared to expression in control tumors. However, exposure to 4000 ppm significantly decreased expression of phospho-Akt (Ser473/Thr308) as compared to the control, indicating a role in the anti-tumorigenic effects of MGD. These findings suggest that MGD may be useful for adjuvant therapy and chemoprevention and that conjugated medium-chain fatty acids may enhance the efficacy of certain chemotherapeutic agents.

Pathological and molecular analyses of atherosclerotic lesions in ApoE-knockout mice.

The establishment of consistent and reliable methods for the analysis of atherosclerosis molecular pathways and for testing the efficiency of new therapeutics is of utmost importance. Here, we fed ApoE-knockout (KO) mice with high-fat diet to for 16 weeks to induce atherosclerosis. Atherosclerotic lesions in mice were methodically investigated using pathologic analyses and molecular biology tools. These lesions were histopathologically classified into three categories: early, progressive, and combined lesions. Immunohistochemical analyses showed that both F4/80 (macrophage marker) and tenascin-C are expressed in these lesions. Real-time PCR analysis conducted using formalin-fixed paraffin-embedded tissues with atherosclerotic lesions demonstrated an increase in the levels of many inflammatory chemokines, including Cxcl16, while antibody arrays performed using frozen atherosclerotic tissue samples showed elevated TIMP-1 expression. Subsequent immunohistochemical analyses showed that the expression of CXCL16, TIMP-1, MMP-9, MMP-8, and LOX-1 is localized in the atherosclerotic lesions. We confirmed that the expression of these proteins is localized to atherosclerotic lesion, which suggests their roles in the development of the lesions in ApoE-KO mice. Therefore, this mouse model represents an appropriate tool for elucidating molecular mechanisms underlying the development of atherosclerosis, and a model for the evaluation of therapeutic efficiency of novel drugs.

PEGylated polyplex with optimized PEG shielding enhances gene introduction in lungs by minimizing inflammatory responses.

Safety is a critical issue in clinical applications of nonviral gene delivery systems. Safe and effective gene introduction into the lungs was previously achieved using polyplexes from poly(ethyleneglycol) (PEG)-block-polycation [PEG-block-PAsp(DET)] and plasmid DNA (pDNA). Although PEGylated polyplexes appeared to be safe, an excess ratio of polycation to pDNA was needed to obtain sufficient transgene expression, which may cause toxicities shortly after gene introduction. In the present study, we investigated the combined use of two polymers, PEG-block-PAsp(DET) (B) and homo PAsp(DET) (H) across a range of mixing ratios to construct polyplexes. Although transgene expressions following in vitro transfections increased in parallel with increased proportions of H, polyplexes with B/H = 50/50 formulation produced the highest expression level following in vivo intratracheal administration. Higher proportions of H elicited high levels of cytokine induction with significant inflammation as assessed by histopathological examinations. Based on the aggregation behavior of polyplexes in bronchoalveolar lavage fluids (BALFs), we suggested that rapid aggregation of polyplexes in the lung induced acute inflammatory responses, resulting in reduced transgene expression. B/H formulation of polyplex can help to improve gene therapy for the respiratory system because it achieves both effective PEG shielding of polyplexes and functioning of PAsp(DET) polycations to enhance endosomal escape.

Dynamic and static control of the off-target interactions of antisense oligonucleotides using toehold chemistry.

Off-target interactions between antisense oligonucleotides (ASOs) with state-of-the-art modifications and biological components still pose clinical safety liabilities. To mitigate a broad spectrum of off-target interactions and enhance the safety profile of ASO drugs, we here devise a nanoarchitecture named BRace On a THERapeutic aSo (BROTHERS or BRO), which is composed of a standard gapmer ASO paired with a partially complementary peptide nucleic acid (PNA) strand. We show that these non-canonical ASO/PNA hybrids have reduced non-specific protein-binding capacity. The optimization of the structural and thermodynamic characteristics of this duplex system enables the operation of an in vivo toehold-mediated strand displacement (TMSD) reaction, effectively reducing hybridization with RNA off-targets. The optimized BROs dramatically mitigate hepatotoxicity while maintaining the on-target knockdown activity of their parent ASOs in vivo. This technique not only introduces a BRO class of drugs that could have a transformative impact on the extrahepatic delivery of ASOs, but can also help uncover the toxicity mechanism of ASOs.

Antitumor effects of chemically modified miR-143 lipoplexes in a mouse model of pelvic colorectal cancer via myristoylated alanine-rich C kinase substrate downregulation.

Replenishing tumor-suppressor miRNAs (TS-miRNAs) is a potential next-generation nucleic acid-based therapeutic approach. Establishing an effective miRNA delivery system is essential to successful TS-miRNA therapy. To overcome vulnerability to RNA nucleases, we previously developed a chemically modified miRNA143-3p (CM-miR-143). In clinical practice, colorectal cancer (CRC) pelvic recurrence is an occasional challenge following curative resection, requiring a novel therapy because reoperative surgery poses a significant burden to the patient. Hence, we considered the use of CM-miR-143 as an alternative treatment. In this study, we used a mouse model bearing pelvic CRC adjacent to the rectum and investigated the anticancer effects of CM-miR-143 lipoplexes formulated from miRNA and a cationic liposome. Compared with commercial synthetic miR-143, CM-miR-143 lipoplexes accumulated heavily in regions of the pelvic CRC tumor where the blood flow was high. As a result, systemic administration of CM-miR-143 lipoplexes improved animal survival by significantly suppressing pelvic CRC tumors and relieving a lethal bowel obstruction caused by rectal compression. Detailed protein analysis revealed that the myristoylated alanine-rich C kinase is a novel target for CM-miR-143 lipoplexes. Our results suggest that CM-miR-143 is a potential next-generation drug candidate in the treatment of CRC pelvic recurrence.

Alterations in cell cycle and induction of apoptotic cell death in breast cancer cells treated with α-mangostin extracted from mangosteen pericarp.

The development of molecularly targeted drugs has greatly advanced cancer therapy, despite these drugs being associated with some serious problems. Recently, increasing attention has been paid to the anticancer effects of natural products. α-Mangostin, a xanthone isolated from the pericarp of mangosteen fruit, has been shown to induce apoptosis in various cancer cell lines and to exhibit antitumor activity in a mouse mammary cancer model. In this study, we investigated the influence of α-mangostin on apoptosis and cell cycle in the human breast cancer cell line MDA-MB231 (carrying a p53 mutation, and HER2, ER, and PgR negative) in order to elucidate its anticancer mechanisms. In α-mangostin-treated cells, induction of mitochondria-mediated apoptosis was observed. On cell-cycle analysis, G1-phase arrest, increased p21(cip1) expression and decreases in cyclins, cdc(s), CDKs and PCNA were observed. In conclusion, α-mangostin may be useful as a therapeutic agent for breast cancer carrying a p53 mutation and having HER2- and hormone receptor-negative subtypes.

Mammary cancer gene therapy targeting lymphangiogenesis: VEGF-C siRNA and soluble VEGF receptor-2, a splicing variant.

Metastasis contributes significantly to cancer mortality, and the most common pathway of initial dissemination is via the afferent ducts of the lymphatics. Overexpression of vascular endothelial growth factor (VEGF)-C has been associated with lymphangiogenesis and lymph node metastasis in a multitude of human neoplasms, including breast cancers. We recently reported that both VEGF-C siRNA and endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2, a new splicing variant) inhibit VEGF-C function and metastasis in a mouse model of metastatic mammary cancer. Here we briefly review our previous experimental work, specifically targeting tumor lymphangiogenesis, in which metastatic mouse mammary cancers received direct intratumoral injections of either expression vectors VEGF-C siRNA or esVEGFR-2, or the empty plasmid vector, once a week for 6 or 8 weeks, followed by in vivo gene electrotransfer of the injected tumors. Throughout our study, both tumor lymphangiogenesis and the multiplicity of lymph node metastasis were significantly inhibited, with an overall reduction in tumor growth, by both VEGF-C siRNA and esVEGFR-2; further, a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells was observed with both treatments. Thus, therapeutic strategies targeting lymphangiogenesis may have great clinical significance for the treatment of metastatic human breast cancer.

α-Mangostin extracted from the pericarp of the mangosteen (Garcinia mangostana Linn) reduces tumor growth and lymph node metastasis in an immunocompetent xenograft model of metastatic mammary cancer carrying a p53 mutation.

BACKGROUND: The mangosteen fruit has a long history of medicinal use in Chinese and Ayurvedic medicine. Recently, the compound α-mangostin, which is isolated from the pericarp of the fruit, was shown to induce cell death in various types of cancer cells in in vitro studies. This led us to investigate the antitumor growth and antimetastatic activities of α-mangostin in an immunocompetent xenograft model of mouse metastatic mammary cancer having a p53 mutation that induces a metastatic spectrum similar to that seen in human breast cancers. METHODS: Mammary tumors, induced by inoculation of BALB/c mice syngeneic with metastatic BJMC3879luc2 cells, were subsequently treated with α-mangostin at 0, 10 and 20 mg/kg/day using mini-osmotic pumps and histopathologically examined. To investigate the mechanisms of antitumor ability by α-mangostin, in vitro studies were also conducted. RESULTS: Not only were in vivo survival rates significantly higher in the 20 mg/kg/day α-mangostin group versus controls, but both tumor volume and the multiplicity of lymph node metastases were significantly suppressed. Apoptotic levels were significantly increased in the mammary tumors of mice receiving 20 mg/kg/day and were associated with increased expression of active caspase-3 and -9. Other significant effects noted at this dose level were decreased microvessel density and lower numbers of dilated lymphatic vessels containing intraluminal tumor cells in mammary carcinoma tissues. In vitro, α-mangostin induced mitochondria-mediated apoptosis and G1-phase arrest and S-phase suppression in the cell cycle. Since activation by Akt phosphorylation plays a central role in a variety of oncogenic processes, including cell proliferation, anti-apoptotic cell death, angiogenesis and metastasis, we also investigated alterations in Akt phosphorylation induced by α-mangostin treatment both in vitro and in vivo. Quantitative analysis and immunohistochemistry showed that α-mangostin significantly decreased the levels of phospho-Akt-threonine 308 (Thr308), but not serine 473 (Ser473), in both mammary carcinoma cell cultures and mammary carcinoma tissues in vivo. CONCLUSIONS: Since lymph node involvement is the most important prognostic factor in breast cancer patients, the antimetastatic activity of α-mangostin as detected in mammary cancers carrying a p53 mutation in the present study may have specific clinical applications. In addition, α-mangostin may have chemopreventive benefits and/or prove useful as an adjuvant therapy, or as a complementary alternative medicine in the treatment of breast cancer.

The endogenous soluble VEGF receptor-2 isoform suppresses lymph node metastasis in a mouse immunocompetent mammary cancer model.

BACKGROUND: Cancer metastasis contributes significantly to cancer mortality and is facilitated by lymphangiogenesis and angiogenesis. A new splicing variant, endogenous soluble vascular endothelial growth factor receptor-2 (esVEGFR-2) that we recently identified is an endogenous selective inhibitor of lymphangiogenesis. To evaluate the antimetastatic potential of esVEGFR-2, gene therapy with vector expressing esVEGFR-2 (pesVEGFR-2) or endostatin (pEndo) as a positive control was conducted on murine metastatic mammary cancer. METHODS: Syngeneic inoculated metastatic mammary cancers received direct intratumoral injection of pesVEGFR-2, pEndo or pVec as control, once a week for six weeks. In vivo gene electrotransfer was performed on the tumors after each injection. RESULTS: Deaths from metastasis were much lower in the pesVEGFR-2 and pEndo groups than in those of the pVec. Tumor volume was significantly lower in the pesVEGFR-2 and the pEndo groups throughout the study. Multiplicity of lymph node and lung metastatic nodules was significantly suppressed in the pesVEGFR-2 and pEndo groups. Moreover, the total number of overall metastasis including the other organs was also decreased in these groups. However, pesVEGFR-2 was not able to decrease the number of lungs, ovaries, kidneys and adrenals with metastasis as counted by unilateral or bilateral metastasis. The number of CD34+/Lyve-1⁻ blood microvessels was significantly decreased in the pEndo group, while the number of CD34⁻/Lyve-1+ lymphatic vessels was significantly decreased in the pesVEGFR-2 and pEndo groups. In addition, a significant reduction in the number of dilated lymphatic vessels containing intraluminal cancer cells was observed in the pesVEGFR-2 and pEndo groups. Levels of apoptosis were significantly increased in the pEndo group, whereas the rates of cell proliferation were significantly decreased in the pesVEGFR-2 and pEndo groups. CONCLUSIONS: Our data demonstrate that esVEGFR-2 can inhibit mainly lymph node metastasis. The antimetastatic activity of esVEGFR-2 may be of high clinical significance in the treatment of metastatic breast cancer because lymph node involvement is a most important prognostic factor in cancer patients.

Raloxifene inhibits tumor growth and lymph node metastasis in a xenograft model of metastatic mammary cancer.

BACKGROUND: The effects of raloxifene, a novel selective estrogen receptor modulator, were studied in a mouse metastatic mammary cancer model expressing cytoplasmic ERα. METHODS: Mammary tumors, induced by inoculation of syngeneic BALB/c mice with BJMC3879luc2 cells, were subsequently treated with raloxifene at 0, 18 and 27 mg/kg/day using mini-osmotic pumps. RESULTS: In vitro study demonstrated that the ERα in BJMC3879luc2 cells was smaller (between 50 and 64 kDa) than the normal-sized ERα (66 kDa) and showed cytoplasmic localization. A statistically significant but weak estradiol response was observed in this cell line. When BJMC3879luc2 tumors were implanted into mice, the ERα mRNA levels were significantly higher in females than in males. In vitro studies showed that raloxifene induced mitochondria-mediated apoptosis and cell-cycle arrest in the G1-phase and a decrease in the cell population in the S-phase. In animal experiments, tumor volumes were significantly suppressed in the raloxifene-treated groups. The multiplicity of lymph node metastasis was significantly decreased in the 27 mg/kg group. Levels of apoptosis were significantly increased in the raloxifene-treated groups, whereas the levels of DNA synthesis were significantly decreased in these groups. No differences in microvessel density in tumors were observed between the control and raloxifene-treated groups. The numbers of dilated lymphatic vessels containing intraluminal tumor cells were significantly reduced in mammary tumors in the raloxifene-treated groups. The levels of ERα mRNA in mammary tumors tended to be decreased in the raloxifene-treated groups. CONCLUSION: These results suggest that the antimetastatic activity of raloxifene in mammary cancer expressing cytoplasmic ERα may be a crucial finding with clinical applications and that raloxifene may be useful as an adjuvant therapy and for the chemoprevention of breast cancer development.

Urethane-induced Mammary Carcinogenesis Susceptibility in Transgenic Mice Expressing a Dominant-negative TGF-ß Type II Receptor.

BACKGROUND/AIM: Transforming growth factor-ß (TGF-ß) plays dual suppressive and oncogenic roles in mammary carcinogenesis. MATERIALS AND METHODS: To analyze whether TGF-ß exerts suppressive or oncogenic actions on mammary carcinogenesis, transgenic mice overexpressing a dominant-negative mutant type II TGF-ß receptor (TßRII-DNR) driven by the mouse mammary tumor virus (MMTV) promoter were treated with a low dose of urethane, a carcinogen present in fermented food products and alcoholic beverages. RESULTS: Lobular proliferative lesions, showing high ß-casein expression, developed in the mammary glands of TßRII-DNR+/+ mice aged >61 weeks. Compared with wild-type mice, TßRII-DNR+/+ mice administered with urethane showed significant increases in dysplastic hyperplasias and adenocarcinomas of the mammary glands. CONCLUSION: The functional decline of TGF-ß signaling in mammary glands led to a high susceptibility to urethane-induced mammary carcinogenesis. TGF-ß signaling may act as a tumor suppressor during mammary tumor development.

Riluzole induces apoptotic cell death in human prostate cancer cells via endoplasmic reticulum stress.

BACKGROUND: Ion channel modulators have been previously associated with cell proliferation and cell death in human cancer cell lines. MATERIALS AND METHODS: The effects of riluzole, an ion channel modulator, on cell proliferation, apoptosis and the apoptotic pathway in the LNCaP and C4-2 prostate cancer cell lines were investigated. RESULTS: Riluzole inhibited DNA synthesis and increased apoptotic cells in both cell lines. The activities of caspase-3, -8 and -9 were significantly increased, and caspase inhibitors for caspase-3, -8 and -9 significantly rescued the cell viability of both carcinoma cell lines treated with riluzole. However, a change in mitochondrial membrane potential, release of cytochrome c and cleavage of Bid were not observed in the riluzole-treated cells. Riluzole significantly induced elevation of caspase-4 activity, fluorescence indicating cytosolic calcium, and morphological changes in endoplasmic reticulum (ER) as observed by transmission electron microscopy. CONCLUSION: Riluzole induces inhibition of DNA synthesis and apoptotic cell death via ER stress in both the LNCaP and C4-2 prostate cancer cell lines.

Phagocytosis mechanism of apoptotic granulosa cells regulated by milk-fat globule-EGF factor 8.

In the process of ovary sexual maturation, most immature ovarian follicles degrade into atretic follicles accompanied by apoptosis in granulosa cells. Macrophages can recognize apoptotic cells through specific binding with phosphatidylserine (PS), exposed on the surface of apoptotic cells, which is mediated by milk-fat globule-EGF factor 8 (MFG-E8). In the present research, we examined the involvement of the MFG-E8-dependent phagocytosis system in the atretic follicles of developing mouse ovaries. The number of atretic follicles and DNA-fragmented granulosa cells significantly increased in B6C3F1 mice during 2 to 6 weeks. Chromatin-condensed granulosa cells were engulfed by macrophages, which existed in the stroma or atretic follicles, or by neighboring normal granulosa cells. MFG-E8 mRNA increased in ovaries during 2 to 6 weeks, and immunoreactivity of MFG-E8 was detected at the surface of apoptotic cells existing around the antrum. Immunoelectron microscopic study revealed MFG-E8-positive signals on the membrane of apoptotic cells near macrophages, but apoptotic cells engulfed by neighboring granulosa cells showed few signals. Anti-Fas antibody elevated the annexin-V-positive reaction in isolated granulosa cells from 3-week-old mouse ovaries. MFG-E8 seems to act on the phagocytosis of apoptotic granulosa cells via macrophages and contribute to the regression process of atretic follicles.

Expression of delta-like 3 is downregulated by aberrant DNA methylation and histone modification in hepatocellular carcinoma.

Delta-like 3 (DLL3) is a member of the Delta/Serrate/Lag-2 family of ligands for the Notch receptor and plays a role in Notch signaling. We have previously revealed that the expression of DLL3 is silenced by aberrant DNA methylation and that overexpression of DLL3 in the HuH2 hepatocellular carcinoma (HCC) cell line induced apoptosis. In the present study, we first confirmed the methylation of DLL3 in HuH2 cells and analyzed the methylation status of the DLL3 promoter region by bisulfite sequencing. Furthermore, we investigated whether other epigenetic modifications, such as histone acetylation and histone methylation, affected the expression of DLL3. Treatment with the DNA methylation inhibitor, 5-azadeoxycytidine (5-Aza-dC) slightly reactivated DLL3 mRNA expression and bisulfite sequencing revealed that CpG sites in the DLL3 promoter region of the HuH2 cells were densely-methylated. In addition, a significant increase in the expression of DLL3 was observed when the cells were treated with 5-Aza-dC in combination with the histone deacetylase inhibitor trichostatin A. However, an inhibitor of the dimethylation of histone H3 lysine 9 (H3K9me2) or the trimethylation of histone H3 lysine 27 (H3K27me3), modifications that are associated with gene silencing, had no effect on DLL3 reactivation. In combination with the findings from our previous study, these results indicated that DLL3 expression was silenced in HCC cells by DNA methylation and was more readily affected by histone acetylation than histone methylation (H3K9me2 or H3K27me3).