Evidence for altered host genetic factors in KSHV infection and KSHV-related disease development.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), the most common AIDS-related malignancy. It also causes other rare, but certainly underreported, KSHV-associated pathologies, namely primary effusion lymphoma, multicentric Castleman disease and KSHV inflammatory cytokine syndrome. Epidemiology and pathogenicity studies point to the potential for host genetic predisposition to KSHV infection and/or the subsequent development of KSHV-associated pathologies partly explaining the peculiar geographic and population-specific incidence of KSHV and associated pathologies and discrepancies in KSHV exposure and infection and KSHV infection and disease development. This review consolidates the current knowledge of host genetic factors involved in the KSHV-driven pathogenesis. Studies reviewed here indicate a plausible connection between KSHV susceptibility and host genetic factors that affect either viral access to host cells via entry mechanisms or host innate immunity to viral infection. Subsequent to infection, KSHV-associated pathogenesis, reviewed here primarily in the context of KS, is likely influenced by an orchestrated concert of innate immune system interactions, downstream inflammatory pathways and oncogenic mechanisms. The association studies reviewed here point to interesting candidate genes that may prove important in achieving a more nuanced understanding of the pathogenesis and therapeutic targeting of KSHV and associated diseases. Recent studies on host genetic factors suggest numerous candidate genes strongly associated with KSHV infection or subsequent disease development, particularly innate immune system mediators. Taken together, these contribute toward our understanding of the geographic prevalence and population susceptibility to KSHV and KSHV-associated diseases.

The in-vitro effect of gonadotropin-releasing hormones, GnRH-I and GnRH-II, on the motility, vitality and acrosome integrity of Vervet monkey (Chlorocebus aethiops) spermatozoa.

Two isoforms of the gonadotropin-releasing hormone (GnRH), GnRH-I and GnRH-II, are expressed in mammals, and the presence of one or more GnRH-like peptides has been demonstrated in the male reproductive tract. GnRH and its receptors (GnRHR) are present in human and non-human primate testis, prostate, epididymis, seminal vesicle, spermatozoa and seminal human plasma. GnRH-II is site-specific and acts directly in an inhibitory or stimulatory fashion. Previous studies speculated that GnRH-II could disrupt specific sperm processes, such as sperm motility or capacitation and could be utilized as an effective contraceptive agent. Our study aimed to investigate the in-vitro effects of GnRH-I and GnRH-II on Vervet monkey sperm function. Electro-ejaculated semen samples from 10 Vervet monkeys (Chlorocebus aethiops) were used to select motile sperm populations. Sperm aliquots were incubated with GnRH-I and GnRH-II at different concentrations for 1 h, where after sperm motility and kinematic parameters were assessed using the automated Sperm Class Analyser. Additional sperm aliquots were incubated with two 10-amino acid control peptides, a non-related peptide and an inactive peptide to exclude the possible influence on sperm motility from other peptides with a structure similar to GnRH. Additionally, a GnRHR-I antagonist (GnRHR-A), Cetrorelix, was tested to establish its antagonistic capability on GnRH. The effect of selected concentrations of GnRH-I and GnRH-II on sperm vitality and acrosome intactness was also evaluated after 10- and 60 min exposure. Analysis of the percentage total sperm motility revealed that different concentrations for GnRH-I and GnRH-II inhibited sperm motility significantly. While sperm progressiveness was also notably affected and a trend of decreased sperm kinematics were evident, no effect was found on sperm vitality or acrosome intactness. The non-related and inactive peptides had no impact on sperm motility. The GnRHR-A demonstrated no effect on sperm motility and effectively blocked the inhibitory outcome on the motility of both GnRH isoforms. While GnRH-I or GnRH-II at low-dose concentrations resulted in in-vitro inhibition of sperm motility, it appears to have no adverse effects on other sperm functional parameters evaluated. These collective observations possibly indicate an essential role for GnRH in the in-vivo process of sperm selection in the female reproductive tract.

Excitatory GABAergic signalling is associated with benzodiazepine resistance in status epilepticus.

Status epilepticus is defined as a state of unrelenting seizure activity. Generalized convulsive status epilepticus is associated with a rapidly rising mortality rate, and thus constitutes a medical emergency. Benzodiazepines, which act as positive modulators of chloride (Cl-) permeable GABAA receptors, are indicated as first-line treatment, but this is ineffective in many cases. We found that 48% of children presenting with status epilepticus were unresponsive to benzodiazepine treatment, and critically, that the duration of status epilepticus at the time of treatment is an important predictor of non-responsiveness. We therefore investigated the cellular mechanisms that underlie acquired benzodiazepine resistance, using rodent organotypic and acute brain slices. Removing Mg2+ ions leads to an evolving pattern of epileptiform activity, and eventually to a persistent state of repetitive discharges that strongly resembles clinical EEG recordings of status epilepticus. We found that diazepam loses its antiseizure efficacy and conversely exacerbates epileptiform activity during this stage of status epilepticus-like activity. Interestingly, a low concentration of the barbiturate phenobarbital had a similar exacerbating effect on status epilepticus-like activity, while a high concentration of phenobarbital was effective at reducing or preventing epileptiform discharges. We then show that the persistent status epilepticus-like activity is associated with a reduction in GABAA receptor conductance and Cl- extrusion capability. We explored the effect on intraneuronal Cl- using both gramicidin, perforated-patch clamp recordings and Cl- imaging. This showed that during status epilepticus-like activity, reduced Cl- extrusion capacity was further exacerbated by activity-dependent Cl- loading, resulting in a persistently high intraneuronal Cl-. Consistent with these results, we found that optogenetic stimulation of GABAergic interneurons in the status epilepticus-like state, actually enhanced epileptiform activity in a GABAAR dependent manner. Together our findings describe a novel potential mechanism underlying benzodiazepine-resistant status epilepticus, with relevance to how this life-threatening condition should be managed in the clinic.

The Contribution of Kaposi's Sarcoma-Associated Herpesvirus to Mortality in Hospitalized Human Immunodeficiency Virus-Infected Patients Being Investigated for Tuberculosis in South Africa.

BACKGROUND: Despite increasing numbers of human immunodeficiency virus (HIV)-infected South Africans receiving antiretroviral therapy (ART), tuberculosis (TB) remains the leading cause of mortality. Approximately 25% of patients treated for TB have microbiologically unconfirmed diagnoses. We assessed whether elevated Kaposi's sarcoma-associated herpesvirus (KSHV) viral load (VL) contributes to mortality in hospitalized HIV-infected patients investigated for TB. METHODS: Six hundred eighty-two HIV-infected patients admitted to Khayelitsha Hospital, South Africa, were recruited, investigated for TB, and followed for 12 weeks. KSHV serostatus, peripheral blood KSHV-VL, and KSHV-associated clinical correlates were evaluated. RESULTS: Median CD4 count was 62 (range, 0-526) cells/µL; KSHV seropositivity was 30.7% (95% confidence interval [CI], 27%-34%); 5.8% had detectable KSHV-VL (median, 199.1 [range, 13.4-2.2 × 106] copies/106 cells); 22% died. Elevated KSHV-VL was associated with mortality (adjusted odds ratio, 6.5 [95% CI, 1.3-32.4]) in patients without TB or other microbiologically confirmed coinfections (n = 159). Six patients had "possible KSHV-inflammatory cytokine syndrome" (KICS): 5 died, representing significantly worse survival (P < .0001), and 1 patient was diagnosed with KSHV-associated multicentric Castleman disease at autopsy. CONCLUSIONS: Given the association of mortality with elevated KSHV-VL in critically ill HIV-infected patients with suspected but not microbiologically confirmed TB, KSHV-VL and KICS criteria may guide diagnostic and therapeutic evaluation.

The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells.

BACKGROUND: Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. METHODS: Using our fluorescently labelled ajoene analogue called dansyl-ajoene, ajoene's protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to ajoene was performed using Schrödinger and pKa calculations by Epik software. Cytotoxicity of ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. RESULTS: The dominant protein tagged by dansyl-ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that ajoene and dansyl-ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by ajoene. CONCLUSIONS: The garlic-derived phytochemical ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of ajoene in cancer cells.

The Nuclear Translocation of Mitogen-Activated Protein Kinases: Molecular Mechanisms and Use as Novel Therapeutic Target.

The mitogen-activated protein kinase (MAPK) cascades are central signaling pathways that play a central role in the regulation of most stimulated cellular processes including proliferation, differentiation, stress response and apoptosis. Currently 4 such cascades are known, each termed by its downstream MAPK components: the extracellular signal-regulated kinase 1/2 (ERK1/2), cJun-N-terminal kinase (JNK), p38 and ERK5. One of the hallmarks of these cascades is the stimulated nuclear translocation of their MAPK components using distinct mechanisms. ERK1/2 are shuttled into the nucleus by importin7, JNK and p38 by a dimer of importin3 with either importin9 or importin7, and ERK5 by importin-α/ß. Dysregulation of these cascades often results in diseases, including cancer and inflammation, as well as developmental and neurological disorders. Much effort has been invested over the years in developing inhibitors to the MAPK cascades to combat these diseases. Although some inhibitors are already in clinical use or clinical trials, their effects are hampered by development of resistance or adverse side-effects. Recently, our group developed 2 myristoylated peptides: EPE peptide, which inhibits the interaction of ERK1/2 with importin7, and PERY peptide, which prevents JNK/p38 interaction with either importin7 or importin9. These peptides block the nuclear translocation of their corresponding kinases, resulting in prevention of several cancers, while the PERY peptide also inhibits inflammation-induced diseases. These peptides provide a proof of concept for the use of the nuclear translocation of MAPKs as therapeutic targets for cancer and/or inflammation.

Vimentin Modulates Infectious Internalization of Human Papillomavirus 16 Pseudovirions.

Human papillomavirus (HPV) infection is the most common viral infection of the reproductive tract, with virtually all cases of cervical cancer being attributable to infection by oncogenic HPVs. However, the exact mechanism and receptors used by HPV to infect epithelial cells are controversial. The current entry model suggests that HPV initially attaches to heparan sulfate proteoglycans (HSPGs) at the cell surface, followed by conformational changes, cleavage by furin convertase, and subsequent transfer of the virus to an as-yet-unidentified high-affinity receptor. In line with this model, we established an in vitro infection system using the HSPG-deficient cell line pgsD677 together with HPV16 pseudovirions (HPV16-PsVs). While pgsD677 cells were nonpermissive for untreated HPV16-PsVs, furin cleavage of the particles led to a substantial increase in infection. Biochemical pulldown assays followed by mass spectrometry analysis showed that furin-precleaved HPV16-PsVs specifically interacted with surface-expressed vimentin on pgsD677 cells. We further demonstrated that both furin-precleaved and uncleaved HPV16-PsVs colocalized with surface-expressed vimentin on pgsD677, HeLa, HaCaT, and NIKS cells, while binding of incoming viral particles to soluble vimentin protein before infection led to a substantial decrease in viral uptake. Interestingly, decreasing cell surface vimentin by small interfering RNA (siRNA) knockdown in HeLa and NIKS cells significantly increased HPV16-PsV infectious internalization, while overexpression of vimentin had the opposite effect. The identification of vimentin as an HPV restriction factor enhances our understanding of the initial steps of HPV-host interaction and may lay the basis for the design of novel antiviral drugs preventing HPV internalization into epithelial cells.IMPORTANCE Despite HPV being a highly prevalent sexually transmitted virus causing significant disease burden worldwide, particularly cancer of the cervix, cell surface events preceding oncogenic HPV internalization are poorly understood. We herein describe the identification of surface-expressed vimentin as a novel molecule not previously implicated in the infectious internalization of HPV16. Contrary to our expectations, vimentin was found to act not as a receptor but rather as a restriction factor dampening the initial steps of HPV16 infection. These results importantly contribute to our current understanding of the molecular events during the infectious internalization of HPV16 and open a new direction in the development of alternative drugs to prevent HPV infection.

Exercise intervention alters HDL subclass distribution and function in obese women.

BACKGROUND: Obesity is associated with a change in high-density lipoprotein (HDL) function and subclass. Exercise training reduces cardiovascular risk in obese patients. We aimed to explore the effect of an exercise training stimulus on HDL functionality and subclass in obese women. METHODS: Thirty-two obese black South African women were randomly assigned to exercise (combined aerobic and resistance exercise) or control (no exercise) conditions for 12-weeks. Pre- and post-testing included venous blood sampling for analysis of lipid profile and HDL functionality, by measuring cellular cholesterol efflux capacity, reduction in endothelial vascular cell adhesion molecule (VCAM) expression (anti-inflammatory function), paraoxonase (PON) (antioxidative function) and platelet activating factor acetylhydrolase (PAF-AH) activities (anti-thrombotic function). PON-1 and PAF-AH expression were determined in serum and in isolated HDL using Western blotting. Levels of large, intermediate and small HDL subclasses were measured using the Lipoprint® system. RESULTS: Exercise training resulted in a decrease in body mass index (- 1.0 ± 0.5% vs + 1.2 ± 0.6%, p = 0.010), PON activity (- 8.7 ± 2.4% vs + 1.1 ± 3.0%, p = 0.021), PAF-AH serum expression (- 22.1 ± 8.0% vs + 16.9 ± 9.8, p = 0.002), and the distribution of small HDL subclasses (- 10.1 ± 5.4% vs + 15.7 ± 6.6%, p = 0.004) compared to controls. Exercise did not alter HDL cellular cholesterol efflux capacity and anti-inflammatory function. CONCLUSIONS: These results demonstrate the potential for exercise training to modify HDL subclass distribution and HDL function in obese women. TRIAL REGISTRATION: Clinical trials number: PACTR201711002789113 .

The Cytotoxicity of the Ajoene Analogue BisPMB in WHCO1 Oesophageal Cancer Cells Is Mediated by CHOP/GADD153.

Garlic is a food and medicinal plant that has been used in folk medicine since ancient times for its beneficial health effects, which include protection against cancer. Crushed garlic cloves contain an array of small sulfur-rich compounds such as ajoene. Ajoene is able to interfere with biological processes and is cytotoxic to cancer cells in the low micromolar range. BisPMB is a synthetic ajoene analogue that has been shown in our laboratory to have superior cytotoxicity to ajoene. In the current study we have performed a DNA microarray analysis of bisPMB-treated WHCO1 oesophageal cancer cells to identify pathways and processes that are affected by bisPMB. The most significantly enriched biological pathways as assessed by gene ontology, KEGG and ingenuity pathway analysis were those involving protein processing in the endoplasmic reticulum (ER) and the unfolded protein response. In support of these pathways, bisPMB was found to inhibit global protein synthesis and lead to increased levels of ubiquitinated proteins. BisPMB also induced alternate splicing of the transcription factor XBP-1; increased the expression of the ER stress sensor GRP78 and induced expression of the ER stress marker CHOP/GADD153. CHOP expression was found to be central to the cytotoxicity of bisPMB as its silencing with siRNA rendered the cells resistant to bisPMB. The MAPK proteins, JNK and ERK1/2 were activated following bisPMB treatment. However JNK activation was not critical in the cytotoxicity of bisPMB, and ERK1/2 activation was found to play a pro-survival role. Overall the ajoene analogue bisPMB appears to induce cytotoxicity in WHCO1 cells by activating the unfolded protein response through CHOP/GADD153.

The garlic compound ajoene targets protein folding in the endoplasmic reticulum of cancer cells.

Ajoene is a natural allylsulfur compound found in crushed garlic that arrests growth and induces apoptosis in cancer cells. To gain mechanistic insights into the cytotoxicity of ajoene in cancer cells, two fluorescently labelled ajoene analogs with dansyl- (DP) and fluorescein- (FOX) tags were synthesized. The tagged ajoenes were found to retain their activity at inhibiting proliferation and inducing apoptosis in MDA-MB-231 human breast-cancer and WHCO1 human esophageal-cancer cells. Both tagged ajoenes localized to the endoplasmic reticulum (ER) in MDA-MB-231 cells as observed by live cell confocal laser scanning microscopy (CLSM) and confirmed by generating an MDA-MB-231 cell line expressing yellow fluorescent protein (YFP) in the ER. DP appears to S-thiolate multiple protein targets in MDA-MB-231 cells as observed by immunoblotting under non-reducing conditions only; and a competition assay demonstrated that DP and Z-ajoene in fact share the same target. Ajoene S-thiolation interfered with protein folding and led to an accumulation of misfolded protein aggregates and activated the unfolded protein response (UPR). Consistent with this mechanism, increased levels of GRP78 and total ubiquitinated proteins were observed; and an ER-folded protein, type-1 collagen, was tracked to the proteasome following ajoene treatment. The intracellular protein aggregates were observed by CLSM and transmission electron microscopy (TEM). This is the first time that ajoene has been shown to target protein folding in the ER of cancer cells. © 2015 Wiley Periodicals, Inc.

Genetic diversity of HHV8 subtypes in South Africa: A5 subtype is associated with extensive disease in AIDS-KS.

Human herpes virus 8 (HHV8) is the etiological agent of all forms of Kaposi's sarcoma (KS). Six major subtypes (A-F), based on genetic variability of open reading frame (ORF)-K1, have been identified. Numerous studies point to differing tumorigenic and pathogenic properties of the HHV8 subtypes. The study objectives were to determine the HHV8 subtypes and their prevalence in a cohort of clinical and histologically confirmed KS in Cape Town, South Africa, and analyze associations between the different subtypes and clinical presentation of KS. Clinical records were prospectively reviewed to extract clinical presentation; demographic data were retrospectively collected and tissue biopsies were taken for ORF-K1 subtyping. Eighty six patients were subtyped; 81 AIDS (acquired immune deficiency syndrome)-KS and 5 African endemic-KS. Subtype A5 (42/86) and B2 (16/86) predominated. B1, B3, A1 and A4 subtypes were identified in 10/86, 9/86, 4/86 and 1/86 patients, respectively. A5 and B subtypes were found in African blacks and individuals of mixed ancestry, while subtypes A1 and A4 were found only in whites and individuals of mixed ancestry. Subtype A5 was associated with >10 KS lesions at presentation in the AIDS cohort (adjusted OR: 3.13; CI: 1.02-9.58). Subtypes A1 and A4 combined were less likely to be associated with poor risk tumor extension (P = 0.031) and A1 was associated with lower likelihood of lower limb involvement (P = 0.019). In conclusion, these results indicate that subtype A5 and B predominate in South Africa and A5 may be associated with more extensive disease.

A novel role of annexin A2 in human type I collagen gene expression.

The fibrillar collagen scaffold of the extracellular matrix provides a structural framework for cells in tissues and regulates intercellular communication; its disregulation has been associated with tumour development and progression. Previous work has shown that expression of type I collagen, the most abundant mammalian extracellular matrix protein, is decreased in chemically or virally transformed cells. This negative regulation could be mapped to a proximal COL1A2 promoter element spanning a CME (Collagen Modulating Element) site in SV40-transformed human fibroblasts (SV-WI38) that binds an unknown repressing protein. By magnetic bead pull-down, we observed a multi-protein complex bound to the CME with preference for single-stranded over conventional double-stranded DNA. MALDI-TOF mass spectrometry of the CME-binding protein complex revealed involvement of nuclear annexin A2 (AnxA2) which was increased in SV40-transformed cells. Further EMSA analysis demonstrated that AnxA2 did not directly bind to the DNA but stabilised the complex and led to an increase in protein binding to the CME in SV-WI38 but not untransformed WI38 cells. Knockdown of AnxA2 by siRNA increased type I collagen production in both WI38 and SV-WI38 cells; however, these effects were not mediated at the transcriptional level. Rather, our data indicate a novel functional role of AnxA2 in the negative post-transcriptional regulation of type I collagen synthesis in human fibroblasts. In SV40-transformed cells, AnxA2 is accumulated at the proximal COL1A2 promoter region, suggesting close association with the transcriptional machinery that possibly facilitates binding to the emerging mRNA, eventually contributing to overall repression of type I collagen protein synthesis.

CCR5 expression is elevated in cervical cancer cells and is up-regulated by seminal plasma.

The interplay between inflammation, cervical cancer and HIV acquisition in women is poorly understood. We have previously shown that seminal plasma (SP) can promote cervical tumour cell growth in vitro and in vivo via the activation of potent inflammatory pathways. In this study, we investigated whether SP could regulate expression of chemokine receptors with known roles in HIV infection, in the cervix and in cervical cancer. The expression of CD4 and CCR5 was investigated by RT-PCR analysis and immunohistochemistry. CD4 and CCR5 expression was elevated in cervical cancer tissue compared with normal cervix. Ex vivo studies conducted on cervical tissues and HeLa cells showed that SP significantly increases the expression of CD4 and CCR5 transcripts. Furthermore, it was found that SP also up-regulates CCR5 protein in HeLa cells. The regulation of CCR5 expression was investigated following treatment of HeLa cells with SP in the presence/absence of chemical inhibitors of intracellular signalling, EP2 and EP4 antagonists, prostaglandin (PG) E2 and a cyclooxygenase (COX)-1 doxycycline-inducible expression system. These experiments demonstrated that the regulation of CCR5 expression by SP occurs via the epidermal growth factor receptor (EGFR)-COX-1-PGE2 pathway. This study provides a link between activation of inflammatory pathways and regulation of HIV receptor expression in cervical cancer cells.

Effects of individual drug and combination antiretroviral therapy on trophoblast proliferation.

BACKGROUND: Combination antiretroviral therapy (cART) has been reported to reduce perinatal transmission of human immunodeficiency virus (HIV) and improve maternal survival outcomes. Recent studies have associated in-utero exposure to cART drugs with adverse outcomes such as pre-eclampsia, preterm delivery, low birth weight and small-for-gestational-age births. However, the exact molecular mechanisms underlying cART-induced adverse pregnancy outcomes remain poorly defined. OBJECTIVES: To investigate the effects of cART drugs on trophoblast proliferation in the HTR-8/SVneo cell line. STUDY DESIGN: HTR-8/SVneo cells were exposed to tenofovir (0.983-9.83 µM), emtricitabine (0.809-8.09 µM) and efavirenz (0.19-1.09 µM), the individual drugs of the first-line single tablet cART regimen termed 'Atripla', and zidovudine (1.12-1.12 µM), lamivudine (0.65-6.5 µM), lopinavir (0.32-3.2 µM) and ritonavir (0.69-6.9 µM), the individual drugs of the second-line single tablet cART regimen termed 'Aluvia'. The cells were treated for 24, 48, 72 and 96 h, and trophoblast proliferation was assessed using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltretrazolium bromide assay. RESULTS: Two-way analysis of variance showed a significant dose-dependent decrease (p < 0.05) in trophoblast proliferation in response to individual and combined drug components of first- and second-line antiretroviral therapy. CONCLUSIONS: First- and second-line cART drugs inhibit trophoblast proliferation, and may contribute to placenta-mediated adverse pregnancy outcomes in patients with HIV.

Seminal plasma induces angiogenic chemokine expression in cervical cancer cells and regulates vascular function.

Cervical cancer is one of the leading gynecological malignancies in women. We have recently shown that seminal plasma (SP) can regulate the inflammatory cyclooxygenase-prostaglandin pathway and enhance the growth of cervical epithelial tumours in vivo by promoting cellular proliferation and alteration of vascular function. This study investigated the molecular mechanism whereby SP regulates vascular function using an in vitro model system of HeLa cervical adenocarcinoma cells and human umbilical vein endothelial cells (HUVECs). We found that SP rapidly enhanced the expression of the angiogenic chemokines, interleukin (IL)-8 and growth regulated oncogene alpha (GRO) in HeLa cells in a time-dependent manner. We investigated the molecular mechanism of SP-mediated regulation of IL-8 and GRO using a panel of chemical inhibitors of cell signalling. We found that treatment of HeLa cells with SP elevated expression of IL-8 and GRO by transactivation of the epidermal growth factor receptor, activation of extracellular signal-regulated kinase and induction of cyclooxygenase enzymes and nuclear factor kappa B. We investigated the impact of IL-8 and GRO, released from HeLa cells after treatment with SP, on vascular function using a co-culture model system of conditioned medium (CM) from HeLa cells, treated with or without SP, and HUVECs. We found that CM from HeLa cells induced the arrangement of endothelial cells into a network of tube-like structures via the CXCR2 receptor on HUVECs. Taken together our data outline a molecular mechanism whereby SP can alter vascular function in cervical cancers via the pro-angiogenic chemokines, IL-8 and GRO.

TBX3 Promotes Cervical Cancer Proliferation and Migration via HPV E6 and E7 Signaling.

Cervical cancer is a leading cause of cancer-related deaths in women globally and 99% of cases are caused by persistent infection with high-risk strains of the human papillomavirus (HPV). The HPV oncoproteins E6 and E7 establish the cancer phenotype by cooperating with host proteins and identifying them may have important therapeutic benefits. T-box transcription factor 3 (TBX3) is a critical developmental regulator, and when it is overexpressed postnatally, it contributes to several cancers, but little is known about its expression and role in cervical cancer. The current study shows that TBX3 is upregulated in cervical cancer cell lines as well as precancerous and cervical cancer patient tissue and is associated with larger and more invasive tumors. Knockdown and overexpression cell culture models show that TBX3 promotes HPV-positive cell proliferation, migration, and spheroid growth; however, TBX3 inhibits these processes in HPV-negative cells. Importantly, we show that the tumor promoting activity of TBX3 in cervical cancer is dependent on E6/E7. IMPLICATIONS: In summary, our study highlights the importance of TBX3 as a cooperating partner of E6/E7 in HPV-positive cervical cancer and identifies TBX3 as a potential therapeutic target to treat this neoplasm.

Activity-Based Proteomic Identification of the S-Thiolation Targets of Ajoene in MDA-MB-231 Breast Cancer Cells.

Garlic is a medicinal plant and spice that has been used for millennia for its health-promoting effects. These medicinal properties are associated with low molecular weight organosulfur compounds, produced following the crushing of garlic cloves. One of these compounds, ajoene, is proposed to act by S-thioallylating cysteine residues on target proteins whose identification in cancer cells holds great promise for understanding mechanistic aspects of ajoene's cancer cell cytotoxicity. To this end, an ajoene analogue (called biotin-ajoene, BA), containing a biotin affinity tag, was designed as an activity-based probe specific for the protein targets of ajoene in MDA-MB-231 breast cancer cells. BA was synthesized via a convergent "click" strategy and found to retain its cytotoxicity against MDA-MB-231 cells compared to ajoene. Widespread biotinylation of proteins was found to occur via disulfide bond formation in a dose-dependent manner, and the biotin-ajoene probe was found to share the same protein targets as its parent compound, ajoene. The biotinylated proteins were affinity-purified from the treated MDA-MB-231 cell lysate using streptavidin-coated magnetic beads followed by an on-bead reduction, alkylation, and digestion to liberate the peptide fragments, which were analyzed by liquid chromatography tandem mass chromatography. A total of 600 protein targets were identified, among which 91% overlapped with proteins with known protein cysteine modification (PCM) sites. The specific sites were enriched for those susceptible to S-glutathionylation (-SSG) (16%), S-sulfhydration (-SSH) (20%), S-sulfenylation (-SOH) (22%), and S-nitrosylation (-SNO) (31%). As target validation, both ajoene and a dansylated ajoene (DP) were found to S-thiolate the pure recombinant forms of glutathione S-transferase pi 1 (GSTP1) and protein disulfide isomerase (PDI), and the ajoene analogue DP was found to be a more potent inhibitor than 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Pathway analysis elucidated that ajoene targets functional and signaling pathways that are implicated in cancer cell survival, specifically cellular processes, metabolism, and genetic information processing pathways. The results of this study provide mechanistic insights into the character of the anti-cancer activity of the natural dietary compound ajoene.

Exogenous Vimentin Supplementation Transiently Affects Early Steps during HPV16 Pseudovirus Infection.

Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus' interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin's stabilisation and formulation before its development as an alternative prophylactic means.

The Garlic Compound Z-Ajoene, S-Thiolates COX2 and STAT3 and Dampens the Inflammatory Response in RAW264.7 Macrophages.

SCOPE: Garlic (Allium sativum) has been used for centuries as a prophylactic and therapeutic medicinal agent to control inflammation-associated pathologies. To investigate the underlying mechanisms, an in vitro inflammatory model is established using RAW264.7 murine macrophages exposed to low-doses of lipopolysaccharide (LPS) in the presence of garlic compounds allicin and Z-ajoene (ZA), mimicking regular garlic consumption. METHODS AND RESULTS: Both allicin and Z-ajoene dampen both transcript and protein expression of the pro-inflammatory cytokines IL1ß, IL6, and IL12ß, and upregulate the expression of the anti-inflammatory cytokine IL10. Protein arrays of selected secreted inflammatory mediators confirm that Z-ajoene has a pronounced down-regulatory effect on LPS-induced inflammatory cytokines and chemokines. Many of these proteins are known targets of the transcription factor signal transducer and activator of transcription 3 (STAT3); and indeed, Z-ajoene or its analogue dansyl-ajoene is found to decrease phosphorylation and nuclear translocation of STAT3, and to covalently modify the protein by S-thiolation at Cys108, Cys367, and Cys687. Z-Ajoene dose-dependently and non-competitively inhibit the activity of cyclooxygenase 2 (COX2), possibly attributed to S-thiolation at Cys9 and Cys299. CONCLUSION: The characterization of Z-ajoene's activity of targeting and covalently modifying STAT3 and COX2, both important regulators of inflammation, may contribute to the health benefits of regular dietary garlic consumption.

Retention and silencing of prepro-GnRH-II and type II GnRH receptor genes in mammals.

The decapeptide hypothalamic-pituitary gonadotrophin-releasing hormone (GnRH)-I and the type I GnRH receptor drive the reproductive hormonal cascade in mammals by stimulating synthesis and secretion of luteinising hormone (LH) and follicle stimulating hormone (FSH). Mammals possess a second GnRH system composed of a related hormone, GnRH-II (differing from GnRH-I by three amino acid residues), and the type II GnRH receptor. In many mammalian species, one or both of the GnRH-II system genes are disrupted or deleted, rendering their products non-functional. This includes humans who possess a gene encoding GnRH-II but lack a functional type II GnRH receptor. Here we examined the genes encoding prepro-GnRH-II (GnRH2) and the type II GnRH receptor (GnRHR2) in more than 20 mammalian species, encompassing 10 orders, to determine whether they encode functional proteins. The structural organisation of both genes in most mammalian genome sequence assemblies was poorly annotated or incompletely described. Our findings show significant variation in the DNA sequence conservation and functional status of each gene, even between closely related species. Prepro-GnRH-II was functionally compromised in 12/22 species and the type II GnRH receptor gene was disrupted in 14/22 species. Retention of large sections of each gene in most mammalian genomes suggests that mammalian ancestors had a functional GnRH-II system. Gene disruptions were due to a spectrum of mutations which must have occurred independently after the evolutionary divergence of mammals from ancestral animals. The genetic information will be useful for understanding the physiological role of the GnRH-II system and establishing animal models for functional studies.