Mehboob Ali

DHA suppresses chronic apoptosis in the lung caused by perinatal inflammation

We have previously shown that an adverse perinatal environment significantly alters lung growth and development and results in persistently altered cardiopulmonary physiology in adulthood. Our model of maternal LPS treatment followed by 14 days of neonatal hyperoxia exposure causes severe pulmonary disease characterized by permanent decreases in alveolarization and diffuse interstitial fibrosis. The current investigations tested the hypothesis that dysregulation of Notch signaling pathways contributes to the permanently altered lung phenotype in our model and that the improvements we have observed previously with maternal docosahexaenoic acid (DHA) supplementation are mediated through normalization of Notch-related protein expression. Results indicated that inflammation (IL-6 levels) and oxidation (F2a-isoprostanes) persisted through 8 wk of life in mice exposed to LPS/O2 perinatally. These changes were attenuated by maternal DHA supplementation. Modest but inconsistent differences were observed in Notch-pathway proteins Jagged 1, DLL 1, PEN2, and presenilin-2. We detected substantial increases in markers of apoptosis including PARP-1, APAF-1, caspase-9, BCL2, and HMGB1, and these increases were attenuated in mice that were nursed by DHA-supplemented dams during the perinatal period. Although Notch signaling is not significantly altered at 8 wk of age in mice with perinatal exposure to LPS/O2, our findings indicate that persistent apoptosis continues to occur at 8 wk of age. We speculate that ongoing apoptosis may contribute to persistently altered lung development and may further enhance susceptibility to additional pulmonary disease. Finally, we found that maternal DHA supplementation prevented sustained inflammation, oxidation, and apoptosis in our model.

Arsenic trioxide induces apoptosis and inhibits the growth of human liver cancer cells

Aims: As a fifth most common cancer type, Hepatocellularcarcinoma (HCC) ranked third leading cause of cancer deaths worldwide. Arsenic trioxide (As2O3) is known as chemotherapeutic agent against few cancer including Acute promyelocyticleukemia and solid tumors. But its effect and possible associated mechanism in HCC is meager. Present study aimed to assess As2O3 modulatory effect on liver cancer by assessing cell growth and viability. Methods: Liver normal (Chang liver) and cancerous cells (Hep3B) were exposed to different concentrations (0, 1, 5, 10 & 15 &mgr;M) of As2O3 at different intervals (24, 48 & 72 h). Cell growth was assessed microscopically, and Cytotoxicity assays were done through 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and Water‐soluble tetrazolium salt (WST) growth inhibition assays. Cell viability was studied by trypan blue staining. Apoptosis was analyzed by Annexin V/PI assay, and expression of genes (Notch and anti‐apoptotic) were determined through western blotting and Q‐PCR method. Key findings: A significant reduction in cell growth and viability was reported in liver cancerous cells as compare to normal cells at 5 &mgr;M As2O3. Consistently, As2O3 induced apoptosis along with down‐regulation of anti‐apoptotic protein Bcl‐xL, and up regulates expression of Notch that leads towards apoptosis. Significance: Results clearly suggest that As2O3 restricted growth and induces apoptosis more in liver cancer cells as compared to normal cells. This finding suggests that it could be a promising potential therapeutic agent against liver cancer which need further testing by in‐vivo investigations.

Alterative Expression and Localization of Profilin 1/VASPpS157 and Cofilin 1/VASPpS239 Regulates Metastatic Growth and Is Modified by DHA Supplementation

Profilin 1, cofilin 1, and vasodialator-stimulated phosphoprotein (VASP) are actin-binding proteins (ABP) that regulate actin remodeling and facilitate cancer cell metastases. miR-17-92 is highly expressed in metastatic tumors and profilin1 and cofilin1 are predicted targets. Docosahexaenoic acid (DHA) inhibits cancer cell proliferation and adhesion. These studies tested the hypothesis that the metastatic phenotype is driven by changes in ABPs including alternative phosphorylation and/or changes in subcellular localization. In addition, we tested the efficacy of DHA supplementation to attenuate or inhibit these changes. Human lung cancer tissue sections were analyzed for F-actin content and expression and cellular localization of profilin1, cofilin1, and VASP (S157 or S239 phosphorylation). The metastatic phenotype was investigated in A549 and MLE12 cells lines using 8 Br-cAMP as a metastasis inducer and DHA as a therapeutic agent. Migration was assessed by wound assay and expression measured by Western blot and confocal analysis. miR-17-92 expression was measured by qRT-PCR. Results indicated increased expression and altered cellular distribution of profilin1/VASPpS157, but no changes in cofilin1/VASPpS239 in the human malignant tissues compared with normal tissues. In A549 and MLE12 cells, the expression patterns of profilin1/VASPpS157 or cofilin1/VASPpS239 suggested an interaction in regulation of actin dynamics. Furthermore, DHA inhibited cancer cell migration and viability, ABP expression and cellular localization, and modulated expression of miR-17-92 in A549 cells with minimal effects in MLE12 cells. Further investigations are warranted to understand ABP interactions, changes in cellular localization, regulation by miR-17-92, and DHA as a novel therapeutic. Mol Cancer Ther; 15(9); 2220–31. ©2016 AACR.

DHA-mediated regulation of lung cancer cell migration is not directly associated with Gelsolin or Vimentin expression

AIMS Deaths associated with cancer metastasis have steadily increased making the need for newer, anti-metastatic therapeutics imparative. Gelsolin and vimentin, actin binding proteins expressed in metastatic tumors, participate in actin remodelling and regulate cell migration. Docosahexaenoic acid (DHA) limits cancer cell proliferation and adhesion but the mechanisms involved in reducing metastatic phenotypes are unknown. We aimed to investigate the effects of DHA on gelsolin and vimentin expression, and ultimately cell migration and proliferation, in this context. MAIN METHODS Non-invasive lung epithelial cells (MLE12) and invasive lung cancer cells (A549) were treated with DHA (30μmol/ml) or/and 8 bromo-cyclic adenosine monophosphate (8 Br-cAMP) (300μmol/ml) for 6 or 24h either before (pre-treatment) or after (post-treatment) plating in transwells. Migration was assessed by the number of cells that progressed through the transwell. Gelsolin and vimentin expression were measured by Western blot and confocal microscopy in cells, and by immunohistochemistry in human lung cancer biopsy samples. KEY FINDINGS A significant decrease in cell migration was detected for A549 cells treated with DHA verses control but this same decrease was not seen in MLE12 cells. DHA and 8 Br-cAMP altered gelsolin and vimentin expression but no clear pattern of change was observed. Immunofluorescence staining indicated slightly higher vimentin expression in human lung tissue that was malignant compared to control. SIGNIFICANCE Collectively, our data indicate that DHA inhibits cancer cell migration and further suggests that vimentin and gelsolin may play secondary roles in cancer cell migration and proliferation, but are not the primary regulators.

Role of SM22 in the differential regulation of phasic vs. tonic smooth muscle

Preliminary proteomics studies between tonic vs. phasic smooth muscles identified three distinct protein spots identified to be those of transgelin (SM22). The latter was found to be distinctly downregulated in the internal anal sphincter (IAS) vs. rectal smooth muscle (RSM) SMC. The major focus of the present studies was to examine the differential molecular control mechanisms by SM22 in the functionality of truly tonic smooth muscle of the IAS vs. the adjoining phasic smooth muscle of the RSM. We monitored SMC lengths before and after incubation with pFLAG-SM22 (for SM22 overexpression), and SM22 small-interfering RNA. pFLAG-SM22 caused concentration-dependent and significantly greater relaxation in the IAS vs. the RSM SMCs. Conversely, temporary silencing of SM22 caused contraction in both types of the SMCs. Further studies revealed a significant reverse relationship between the levels of SM22 phosphorylation and the amount of SM22-actin binding in the IAS and RSM SMC. Data showed higher phospho-SM22 levels and decreased SM22-actin binding in the IAS, and reverse to be the case in the RSM SMCs. Experiments determining the mechanism for SM22 phosphorylation in these smooth muscles revealed that Y-27632 (Rho kinase inhibitor) but not Gö-6850 (protein kinase C inhibitor) caused concentration-dependent decreased phosphorylation of SM22. We speculate that SM22 plays an important role in the regulation of basal tone via Rho kinase-induced phosphorylation of SM22.

Changes in Vasodilator-Stimulated Phosphoprotein Phosphorylation, Profilin-1, and Cofilin-1 in Accreta and Protection by DHA

Accreta and gestational trophoblastic disease (ie, choriocarcinoma) are placental pathologies characterized by hyperproliferative and invasive trophoblasts. Cellular proliferation, migration, and invasion are heavily controlled by actin-binding protein (ABP)-mediated actin dynamics. The ABP vasodilator-stimulated phosphoprotein (VASP) carries key regulatory role. Profilin-1, cofilin-1, and VASP phosphorylated at Ser157 (pVASP-S157) and Ser239 (pVASP-S239) are ABPs that regulate actin polymerization and stabilization and facilitate cell metastases. Docosahexaenoic acid (DHA) inhibits cancer cell migration and proliferation. We hypothesized that analogous to malignant cells, ABPs regulate these processes in extravillous trophoblasts (EVTs), which exhibit aberrant expression in placenta accreta. Placental–myometrial junction biopsies of histologically confirmed placenta accreta had significantly increased immunostaining levels of cofilin-1, VASP, pVASP-S239, and F-actin. Treatment of choriocarcinoma-derived trophoblast (BeWo) cells with DHA (30 µM) for 24 hours significantly suppressed proliferation, migration, and pVASP-S239 levels and altered protein profiles consistent with increased apoptosis. We concluded that in accreta changes in the ABP expression profile were a response to restore homeostasis by counteracting the hyperproliferative and invasive phenotype of the EVT. The observed association between VASP phosphorylation, apoptosis, and trophoblast proliferation and migration suggest that DHA may offer a therapeutic solution for conditions where EVT is hyperinvasive.