The Blood-Testis barrier disruption is a prerequisite for Toxicant-Induced peritubular macrophage increases in the testis of peripubertal rats.

Peritubular macrophages (PTMφ) are predominantly localized near spermatogonial stem cells in the testis. We previously revealed that exposure of peripubertal male Fischer rats to mono-(2-ethylhexyl) phthalate (MEHP) leads to increased PTMφs in the testis. The mechanisms that trigger increases in PTMφs in the testis are poorly understood. However, MEHP exposure is known to both induce spermatocyte apoptosis and to perturb the blood-testis barrier (BTB). This study aims to elucidate the association between the disruption of BTB and the increases of PTMφs in the testis by comparing the effects observed with MEHP to two other testicular toxicants with variable effects on the BTB and subtype of germ cell undergoing apoptosis. Methoxyacetic acid (MAA) acts directly on spermatocytes and does not affect BTB function, while cadmium chloride (CdCl2) induces profound injury to BTB. The results indicated that MAA exposure significantly increased spermatocyte apoptosis, while no significant changes in the numbers of PTMφs in the testis occurred. In contrast, CdCl2 exposure disrupted BTB function and increased the abundance of PTMφs in the testis. To further investigate whether MEHP-induced changes in BTB integrity accounted for the increase in PTMφs, a plasmid for LG3/4/5, the functional component of laminin-alpha 2, was overexpressed in the testis to stabilize BTB integrity before MEHP exposure. The results showed that LG3/4/5 overexpression substantially reduced the ability of MEHP to compromise BTB integrity and prevented the increase in PTMφ numbers after MEHP exposure. These results indicate that BTB disruption is necessary to increase PTMφs in the testis induced by toxicants.

Responses of peritubular macrophages and the testis transcriptome profiles of peripubertal and adult rodents exposed to an acute dose of MEHP.

Exposure of rodents to mono-(2-ethylhexyl) phthalate (MEHP) is known to disrupt the blood-testis barrier and cause testicular germ cell apoptosis. Peritubular macrophages (PTMφ) are a newly identified type of testicular macrophage that aggregates near the spermatogonial stem cell niche. We have previously reported that MEHP exposure increased the numbers of PTMφs by 6-fold within the testis of peripubertal rats. The underlying mechanism(s) accounting for this change in PTMφs and its biological significance is unknown. This study investigates if MEHP-induced alterations in PTMφs occur in rodents (PND 75 adult rats and PND 26 peripubertal mice) that are known to be less sensitive to MEHP-induced testicular toxicity. Results show that adult rats have a 2-fold higher basal level of PTMφ numbers than species-matched peripubertal animals, but there was no significant increase in PTMφ numbers after MEHP exposure. Peripubertal mice have a 5-fold higher basal level of PTMφ compared with peripubertal rats but did not exhibit increases in number after MEHP exposure. Further, the interrogation of the testis transcriptome was profiled from both the MEHP-responsive peripubertal rats and the less sensitive rodents via 3' Tag sequencing. Significant changes in gene expression were observed in peripubertal rats after MEHP exposure. However, adult rats showed lesser changes in gene expression, and peripubertal mice showed only minor changes. Collectively, the data show that PTMφ numbers are associated with the sensitivity of rodents to MEHP in an age- and species-dependent manner.

Mono-(2-ethylhexyl) phthalate (MEHP) reversibly disrupts the blood-testis barrier (BTB) in pubertal rats.

The blood-testis barrier (BTB) is constituted by tight junctions between adjacent Sertoli cells (SC) that create a specialized adluminal microenvironment to foster the development of spermatocytes and spermatids. The BTB is a well-studied target of numerous environmental toxicants, including di-(2-ethylhexyl) phthalate (DEHP), a compound widely used in various consumer products. MEHP is the active toxic metabolite of DEHP that has long been recognized in postnatal rodents to disrupt SC function. This study evaluates the impact of MEHP on the integrity of the BTB in both pubertal and adult rats and the signal transduction pathways known to be involved in the disruption of the BTB. Treatment of prepubertal rats with 700 mg/kg MEHP for 24 hours functionally disrupted the BTB integrity. A similar treatment of adult rats with MEHP did not disrupt the integrity of the BTB. The observed disruption of the BTB integrity in the MEHP-treated prepubertal rats occurred concomitantly with a decreased expression and mislocalization of both the ZO1 and occludin tight junction-associated proteins, as well as sloughing of spermatocytes and spermatids. At this same time, MEHP treatment induced a transient surge of p44/42 mitogen-activated protein kinase (MAPK) pathway. Interestingly, after a recovery period of 5 weeks, the BTB recovered and was functionally intact. This is the first report to indicate that acute MEHP exposure of prepubertal rats, but not adult rats, disrupts the functional integrity of the BTB and that this effect on the BTB is reversible.

Peritubular Macrophages Are Recruited to the Testis of Peripubertal Rats After Mono-(2-Ethylhexyl) Phthalate Exposure and Is Associated With Increases in the Numbers of Spermatogonia.

Peripubertal exposure of male rodents to the phthalate metabolite mono-(2-ethylhexyl) phthalate (MEHP) causes testicular inflammation, spermatocyte apoptosis, and disruption of the blood-testis barrier. The MEHP-induced inflammatory response in the testis includes an infiltration of macrophages and neutrophils, although the cause and purpose of this response is unknown. Recently, a population of testicular macrophages known as peritubular macrophages that are phenotypically distinct from those resident in interstitium was described in mice. Peritubular macrophages aggregate near the spermatogonial stem cell niche and are believed to stimulate their differentiation. We hypothesized that if testicular peritubular macrophages do indeed stimulate spermatogonial differentiation, MEHP exposure would result in an increase of peritubular macrophages to stimulate the replacement of lost spermatocytes. Male rats were exposed to 700 mg/kg MEHP or corn oil (vehicle control) via oral gavage at postnatal day 28 and euthanized at 48 h, 1 or 2 weeks later. Seminiferous tubules were stained with immunofluorescent markers for macrophages (major histocompatibility complex class II [MHC-II+]) and undifferentiated spermatogonia (PLZF). Peritubular macrophages were observed in rat testis: MHC-II+ cells on the surface of seminiferous tubules with heterogeneous morphology. Quantification of MHC-II+ cells revealed that, unlike in the mouse, their numbers did not increase through puberty (2-week period). MEHP increased macrophage presence by 6-fold 48 h after exposure and remained elevated by 2-fold 2 weeks after exposure. An increase of differentiating spermatogonia occurred 2 weeks after MEHP exposure. Taken together, our results suggest that peritubular macrophages play a crucial role in the testis response to acute injury and the subsequent recovery of spermatogenesis.

MEHP-induced rat testicular inflammation does not exacerbate germ cell apoptosis.

The testis is an organ that maintains an immune suppressive environment. We previously revealed that exposure of pre-pubertal rats to an acute dose of a well-described Sertoli cell toxicant, mono-(2-ethylhexyl) phthalate (MEHP), leads to an accumulation of CD11b+ immune cells in the testicular interstitial space that closely correlates with a robust incidence of germ cell (GC) apoptosis. Here, we test the hypothesis that the infiltrating immune cells contribute to GC apoptosis. Postnatal day 28 Fischer rats that received an oral dose of 700 mg/kg MEHP showed a significant infiltration of both CD11bc+/CD68+/CD163- macrophages and neutrophils. The infiltration peaked at 12 h, but had reduced by 48 h. Testicular macrophages from MEHP-treated rats showed significantly upregulated expression of Tnfa and Il6, and the Arg1/Nos2 ratio was reduced compared to controls. However, small increases in anti-inflammatory genes Il10 and Tgfb1 were also observed. Depletion of circulating monocytes with clodronate liposomes prior to MEHP treatment reduced the macrophage influx into the testis, but did not lower GC apoptosis. Additionally, depletion of neutrophils using an anti-polymorphonuclear cell antibody prevented both macrophage and neutrophil infiltration into the testis, and also did not affect GC apoptosis. Together, these results show that exposure to MEHP leads to a rapid and temporary influx of pro-inflammatory monocytes and neutrophils in the interstitium of the testis. However, with this acute dosing paradigm, these infiltrating leukocytes do not appear to contribute to MEHP-induced testicular GC apoptosis leaving the functional significance of these infiltrating cells in the pathogenesis of MEHP-induced testicular injury unresolved.

Distinct roles of different forms of vitamin E in DHA-induced apoptosis in triple-negative breast cancer cells.

SCOPE: Docosahexaenoic acid (DHA) has been shown to exhibit anticancer actions in vitro and in vivo in a variety of cancers. Here, we investigated the role for DHA in inducing apoptosis in triple-negative breast cancer (TNBC) and studied the mechanisms of action. METHODS AND RESULTS: DHA induces apoptosis as detected by Annexin V-FITC/PI assay as well as induces cleavage of caspase-8 and -9, endoplasmic reticulum stress (ERS), and elevated levels of death receptor-5 (DR5) protein expression as detected by western blot assays. Chemical inhibitors of caspase-8 and -9 and small interfering RNAs (siRNAs) show DHA to induce ERS/CHOP/DR5-mediated caspase-8 and -9 dependent apoptosis. Furthermore, DHA induces elevated cellular levels of reactive oxygen species (ROS) and antioxidant; RRR-α-tocopherol (αT) blocked DHA-induced apoptotic events. In contrast to the antagonistic impact of αT, gamma-tocotrienol (γT3) was demonstrated to cooperate with DHA in inducing apoptotic events in TNBC cells. CONCLUSION: Data, for the first time, demonstrate that DHA induces apoptosis in TNBC cells via activation of ERS/CHOP/DR5-mediated caspase-8 and -9 dependent pro-apoptotic events, and that different forms of vitamin E exhibit distinct effects on DHA-induced apoptosis; namely, inhibition by αT and enhancement by γT3.

Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer.

INTRODUCTION: Adjuvant treatment with tamoxifen substantially improves survival of women with estrogen-receptor positive (ER+) tumors. Tamoxifen resistance (TAMR) limits clinical benefit. RRR-α-tocopherol ether-linked acetic acid analogue (α-TEA) is a small bioactive lipid with potent anticancer activity. We evaluated the ability of α-TEA in the presence of tamoxifen to circumvent TAMR in human breast cancer cell lines. METHODS: Two genotypically matched sets of TAM-sensitive (TAMS) and TAM-resistant (TAMR) human breast cancer cell lines were assessed for signal-transduction events with Western blotting, apoptosis induction with Annexin V-FITC/PI assays, and characterization of cholesterol-rich microdomains with fluorescence staining. Critical involvement of selected mediators was determined by using RNA interference and chemical inhibitors. RESULTS: Growth-factor receptors (total and phosphorylated forms of HER-1 and HER-2), their downstream prosurvival mediators pAkt, pmTOR, and pERK1/2, phosphorylated form of estrogen receptor-α (pER-α at Ser-167 and Ser-118, and cholesterol-rich lipid microdomains were highly amplified in TAMR cell lines and enhanced by treatment with TAM. α-TEA disrupted cholesterol-rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators, and induced DR5-mediated mitochondria-dependent apoptosis via an endoplasmic reticulum stress-triggered pro-death pJNK/CHOP/DR5 amplification loop. Furthermore, methyl-ß-cyclodextrin (MßCD), a chemical disruptor of cholesterol rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators and to induce apoptosis. CONCLUSIONS: Data for the first time document that targeting cholesterol-rich lipid microdomains is a potential strategy to circumvent TAMR, and the combination of α-TEA + TAM can circumvent TAMR by suppression of prosurvival signaling via disruption of cholesterol-rich lipid microdomains and activation of apoptotic pathways via induction of endoplasmic reticulum stress.

α-TEA cooperates with chemotherapeutic agents to induce apoptosis of p53 mutant, triple-negative human breast cancer cells via activating p73.

INTRODUCTION: Successful treatment of p53 mutant, triple-negative breast cancers (TNBC) remains a daunting challenge. Doxorubicin (DOXO) and cisplatin (CDDP) are standard-of-care treatments for TNBC, but eventually fail due to acquired drug resistance and toxicity. New treatments for overcoming drug resistance and toxicity in p53 mutant, TNBC are therefore badly needed. Unlike p53, p73 - a member of the p53 family - is usually not mutated in cancers and has been shown to regulate p53-mediated apoptotic signaling in p53-deficient cancers. Therefore, identification of anticancer agents that can activate p73 in p53-deficient cancers may provide a chemotherapeutic approach for treatment of p53 mutant cancers. Here we report on the reconstitution of the p53 tumor suppressor pathway in a p53-independent manner via p73 with combination treatments of α-TEA, a small bioactive lipid, plus DOXO or CDDP. METHODS: p53 mutant, TNBC cell lines MDA-MB-231, BT-20 and MDA-MB-468 were used to evaluate the anticancer effect of chemotherapeutic drugs and α-TEA using annexin V (FITC)/PI staining, western blot analyses, RT-PCR and siRNA knockdown techniques. RESULTS: Combination treatments of α-TEA plus DOXO or CDDP act cooperatively to induce apoptosis, caspase-8 and caspase-9 cleavage, p73, phospho-c-Ab1 and phospho-JNK protein expression, and increase expression of p53 downstream mediators; namely, death receptor-5, CD95/APO-1 (Fas), Bax and Noxa, as well as Yap nuclear translocation - plus reduce expression of Bcl-2. Knockdown of p73, c-Abl, JNK or Yap using siRNAs shows that p73 plays a critical role in combination treatment-enhanced apoptosis and the expression of pro-apoptotic and anti-apoptotic mediators, and that c-Abl, JNK and Yap are upstream mediators of p73 in combination treatment responses. CONCLUSIONS: Data show that α-TEA in combination with DOXO or CDDP synergistically enhances apoptosis in TNBC via targeting p53-mediated genes in a p73-dependent manner, and that p73 responses are downstream of c-Abl, JNK and Yap.

α-TEA-induced death receptor dependent apoptosis involves activation of acid sphingomyelinase and elevated ceramide-enriched cell surface membranes.

BACKGROUND: Alpha-tocopherol ether-linked acetic acid (α-TEA), an analog of vitamin E (RRR-alpha-tocopherol), is a potent and selective apoptosis-inducing agent for human cancer cells in vivo and in vitro. α-TEA induces apoptosis via activation of extrinsic death receptors Fas (CD95) and DR5, JNK/p73/Noxa pathways, and suppression of anti-apoptotic mediators Akt, ERK, c-FLIP and survivin in breast, ovarian and prostate cancer cells. RESULTS: In this study, we demonstrate that α-TEA induces the accumulation of cell surface membrane ceramide, leading to co-localization with Fas, DR5, and FADD, followed by activation of caspases-8 and -9 and apoptosis in human MDA-MB-231 breast cancer cells. α-TEA treatment leads to increased acid sphingomyelinase (ASMase) activity by 30 min, peaking at 4 hrs, which is correlated with ASMase translocation from cytosol to the cell surface membrane. Functional knockdown of ASMase with either the chemical inhibitor, desipramine, or siRNA markedly reduces α-TEA-induced cell surface membrane accumulation of ceramide and its co-localization with Fas, DR5, and FADD, cleavage of caspases-8 and -9 and apoptosis, suggesting an early and critical role for ASMase in α-TEA-induced apoptosis. Consistent with cell culture data, immunohistochemical analyses of tumor tissues taken from α-TEA treated nude mice bearing MDA-MB-231 xenografts show increased levels of cell surface membrane ceramide in comparison to tumor tissues from control animals. CONCLUSION: Taken together, these studies demonstrate that ASMase activation and membrane ceramide accumulation are early events contributing to α-TEA-induced apoptosis in vitro and perhaps in vivo.

α-TEA induces apoptosis of human breast cancer cells via activation of TRAIL/DR5 death receptor pathway.

Vitamin E derivative RRR-α-tocopherol ether-linked acetic acid analog (α-TEA) induces apoptosis in MCF-7 and HCC-1954 human breast cancer cells in a dose- and time-dependent manner. α-TEA induces increased levels of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and death receptor-5 (DR5) and decreased levels of antiapoptotic factor, cellular FLICE-like inhibitory protein (c-FLIP L). DR5/TRAIL induced apoptosis involves downregulation of c-FLIP (L), caspase-8 activation, activated proapoptotic mediators tBid and Bax, mitochondrial permeability transition, and activation of caspase-9. siRNA knockdown of either DR5 or TRAIL blocks the ability of α-TEA to enhance DR5 protein levels, downregulate c-FLIP(L) protein levels and induce apoptosis. Combination of α-TEA + TRAIL acts cooperatively to induce apoptosis, and increase DR5 and decrease c-FLIP (L) protein levels. siRNA knockdown of c-FLIP produces a low level of spontaneous apoptosis and enhances α-TEA- and TRAIL-induced apoptosis. Taken together, these studies show that α-TEA induces TRAIL/DR5 mitochondria-dependent apoptosis in human breast cancer cells, and that TRAIL/DR5-dependent increases in DR5 and decreases in c-FLIP expression are triggered by TRAIL or α-TEA treatments.

Role of endoplasmic reticulum stress in alpha-TEA mediated TRAIL/DR5 death receptor dependent apoptosis.

BACKGROUND: Alpha-TEA (RRR-alpha-tocopherol ether-linked acetic acid analog), a derivative of RRR-alpha-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in alpha-TEA induced apoptosis in human breast cancer cells. METHODOLOGY/PRINCIPAL FINDINGS: alpha-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2alpha), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) alpha-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii) alpha-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP's inhibition of caspase-8; and (iii) alpha-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling. CONCLUSION: Taken together, ER stress plays an important role in alpha-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling.

RRR-gamma-tocopherol induces human breast cancer cells to undergo apoptosis via death receptor 5 (DR5)-mediated apoptotic signaling.

Goal of this study was to investigate the pro-apoptotic properties of RRR-gamma-tocopherol (gammaT) in human breast cancer cells. gammaT was shown to induce cancer cells but not normal cells to undergo apoptosis, sensitize cancer cells to Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL)-induced apoptosis, and increase death receptor 5 (DR5) mRNA, protein and cell surface expression. Knockdown of DR5 attenuated gammaT-induced apoptosis. Investigations of post-receptor signaling showed: caspase-8, Bid and Bax activation, increases in mitochondria permeability, cytochrome c release and caspase-9 activation. Thus, gammaT is a potent pro-apoptotic agent for human breast cancer cells inducing apoptosis via activation of DR5-mediated apoptotic pathway.