Nagarajarao Shamaladevi

CXC receptor-1 silencing inhibits androgen-independent prostate cancer.

The CXC receptor-1 (CXCR1) is a coreceptor for interleukin-8 (IL-8) and is expressed on both normal and tumor cells. The function of CXCR1 in prostate cancer was investigated by silencing its expression, using RNA interference. We established stable cell colonies of PC-3 cells, depleted of CXCR1, using lentiviral plasmids (pLK0.1puro) generating small hairpin RNA (shRNA) against CXCR1 mRNA. Stable shRNA transfectants (PLK1-PLK5) that express significantly reduced CXCR1 mRNA (>or=90% down) and protein (>or=43% down) or vector-only transfectants (PC-3V) were characterized. PLK cells showed reduced cell proliferation (down, >or=66%), due to cell cycle arrest at G(1)-S phase, decreases in Cyclin D1, CDK4, phosphorylated Rb, and extracellular signal-regulated kinase 1/2 levels compared with those in PC-3V cells. CXCR1 depletion lead to increases in spontaneous apoptosis by mitochondria-mediated intrinsic mechanism and increases in proapoptotic proteins (BAD, 40%; BAX, 12%), but decreases in antiapoptotic proteins (BCL2, down 38%; BCL(xL), 20%). PLK2 cells grew as slow-growing tumors (decrease of 54%), compared with that of PC3V tumors in athymic mice. Ex vivo analyses of PLK2 tumor tissues showed reduced expression of Cyclin D1 and vascular endothelial growth factor, and increased apoptosis activity. Other IL-8-expressing prostate cancer cell lines also exhibited similar phenotypes when CXCR1 was depleted by CXCR1 shRNA transfection. In contrast to these cells, CXCR1 depletion had little effect on IL-8 ligand-deficient LNCaP cells. RNA interference rescue using mutated CXCR1 plasmids reversed the silencing effect of PLK2, thus demonstrating the specificity of phenotypic alteration by CXCR1 shRNA. These studies establish that CXCR1 promotes IL-8-mediated tumor growth.

Decreased Astrocytic Thrombospondin-1 Secretion After Chronic Ammonia Treatment Reduces the Level of Synaptic Proteins: In Vitro and In Vivo Studies

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin‐1 (TSP‐1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5–2.5 mM) for 1–10 days, and TSP‐1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra‐ and extracellular TSP‐1 levels. Exposure of cultured neurons to conditioned media from ammonia‐treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP‐1 over‐expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP‐1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP‐1 synthesis in other cell types, also reversed the ammonia‐induced TSP‐1 reduction. Likewise, we found a significant decline in TSP‐1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP‐1 may represent an important therapeutic target for CHE.

Shrinkage of experimental benign prostatic hyperplasia and reduction of prostatic cell volume by a gastrin-releasing peptide antagonist

Gastrin releasing-peptide (GRP) is a potent growth factor in many malignancies. Benign prostatic hyperplasia (BPH) is a progressive age-related proliferation of glandular and stromal tissues; various growth factors and inflammatory processes are involved in its pathogenesis. We have demonstrated that potent antagonists of GRP inhibit growth of experimental human tumors including prostate cancer, but their effect on models of BPH has not been studied. Here, we evaluated the effects of GRP antagonist RC-3940-II on viability and cell volume of BPH-1 human prostate epithelial cells and WPMY-1 prostate stromal cells in vitro, and in testosterone-induced BPH in Wistar rats in vivo. RC-3940-II inhibited the proliferation of BPH-1 and WPMY-1 cells in a dose-dependent manner and reduced prostatic cell volume in vitro. Shrinkage of prostates was observed after 6 wk of treatment with RC-3940-II: a 15.9% decline with 25 μg/d; and a 18.4% reduction with 50 μg/d (P < 0.05 for all). Significant reduction in levels of proliferating cell nuclear antigen, NF-κβ/p50, cyclooxygenase-2, and androgen receptor was also seen. Analysis of transcript levels of genes related to growth, inflammatory processes, and signal transduction showed significant changes in the expression of more than 90 genes (P < 0.05). In conclusion, GRP antagonists reduce volume of human prostatic cells and lower prostate weight in experimental BPH through direct inhibitory effects on prostatic GRP receptors. GRP antagonists should be considered for further development as therapy for BPH.

Defective synthesis and release of astrocytic thrombospondin-1 mediates the neuronal TDP-43 proteinopathy, resulting in defects in neuronal integrity associated with chronic traumatic encephalopathy: in vitro studies

Transactivating DNA‐binding protein‐43 (TDP‐43) inclusions and the accumulation of phosphorylated and ubiquitinated tau proteins (p‐tau) have been identified in postmortem brain specimens from patients with chronic traumatic encephalopathy (CTE). To examine whether these proteins contribute to the development of CTE, we utilized an in vitro trauma system known to reproduce many of the findings observed in humans and experimental animals with traumatic brain injury. Accordingly, we examined the role of TDP‐43 and Tau in an in vitro model of trauma, and determined whether these proteins contribute to the defective neuronal integrity associated with CNS trauma. Single or multiple episodes of trauma to cultured neurons resulted in a time‐dependent increase in cytosolic levels of phosphorylated TDP‐43 (p‐TDP‐43). Trauma to cultured neurons also caused an increase in levels of casein kinase 1 epsilon (CK1ε), and ubiquitinated p‐TDP‐43, along with a decrease in importin‐β (all factors known to mediate the “TDP‐43 proteinopathy”). Defective neuronal integrity, as evidenced by a reduction in levels of the NR1 subunit of the NMDA receptor, and in PSD95, along with increased levels of phosphorylated tau were also observed. Additionally, increased levels of intra‐ and extracellular thrombospondin‐1 (TSP‐1) (a factor known to regulate neuronal integrity) were observed in cultured astrocytes at early stages of trauma, while at later stages decreased levels were identified. The addition of recombinant TSP‐1, conditioned media from cultured astrocytes at early stages of trauma, or the CK1ε inhibitor PF4800567 hydrochloride to traumatized cultured neurons reduced levels of p‐TDP‐43, and reversed the trauma‐induced decline in NR1 subunit of the NMDA receptor and PSD95 levels. These findings suggest that a trauma‐induced increase in TDP‐43 phosphorylation contributes to defective neuronal integrity, and that increasing TSP‐1 levels may represent a useful therapeutic approach for the prevention of the neuronal TDP‐43 proteinopathy associated with CTE.

The andean anticancer herbal product BIRM causes destabilization of androgen receptor and induces caspase-8 mediated-apoptosis in prostate cancer

BIRM is an anticancer herbal formulation from Ecuador. Previous study established its antitumor and antimetastatic activity against prostate cancer models. The activity of BIRM against human prostate cancer (PCa) cells was investigated to uncover its mechanism of antitumor activity. In androgen receptor (AR)-expressing PCa cells BIRM was 2.5-fold (250%) more cytotoxic in presence of androgen (DHT) compared to cells grown in the absence of DHT. In AR-positive cells (LAPC-4 and LNCaP) BIRM caused a dose and time-dependent down-regulation of AR and increased apoptosis. Exposing cells to BIRM did not affect the synthesis of AR and AR promoter activity but increased degradation of AR via proteasome-pathway. BIRM caused destabilization of HSP90-AR association in LAPC-4 cells. It induced apoptosis in PCa cells by activation of caspase-8 via death receptor and FADD-mediated pathways. A synthetic inhibitor of Caspase-8 cleavage (IETD-CHO) aborted BIRM–induced apoptosis. The effect of BIRM on AKT-mediated survival pathway in both AR+ and AR- negative (PC-3 and DU145) showed decreased levels of p-AKTser 473 in all PCa cell lines. BIRM dosed by oral gavage in mice bearing PC-3ML tumors showed selective efficacy on tumor growth; before tumors are established but limited efficacy when treated on existing tumors. Moreover, BIRM inhibited the LNCaP tumor generated by orthotropic implantation into dorsal prostate of nude mice. Partial purification of BIRM by liquid-liquid extraction and further fractionation by HPLC showed 4-fold increased specific activity on PCa cells. These results demonstrate a mechanistic basis of anti-tumor activity of the herbal extract BIRM.

Differential Response of Neural Cells to Trauma-Induced Swelling In Vitro

Brain edema and the associated increase in intracranial pressure are major consequences of traumatic brain injury (TBI) that accounts for most early deaths after TBI. We recently showed that acute severe trauma to cultured astrocytes results in cell swelling. We further examined whether trauma induces cell swelling in neurons and microglia. We found that severe trauma also caused cell swelling in cultured neurons, whereas no swelling was observed in microglia. While severe trauma caused cell swelling in both astrocytes and neurons, mild trauma to astrocytes, neurons, and microglia failed to cell swelling. Since extracellular levels of glutamate are increased in brain post-TBI and microglia are known to release cytokine, and direct exposure of astrocytes to these molecules are known to stimulate cell swelling, we examined whether glutamate or cytokines have any additive effect on trauma-induced cell swelling. Exposure of cultured astrocytes to trauma caused cell swelling, and such swelling was potentiated by the exposure of traumatized astrocytes to glutamate and cytokines. Conditioned medium (CM) from traumatized astrocytes had no effect on neuronal swelling post-trauma, while CM from traumatized neurons and microglia potentiated the effect of trauma on astrocyte swelling. Further, trauma significantly increased the Na–K–Cl co-transporter (NKCC) activity in neurons, and that inhibition of NKCC activity diminished the trauma-induced neuronal swelling. Our results indicate that a differential sensitivity to trauma-induced cell swelling exists in neural cells and that neurons and microglia are likely to be involved in the potentiation of the astrocyte swelling post-trauma.

Decreased STAT3 Phosphorylation Mediates Cell Swelling in Ammonia-Treated Astrocyte Cultures

Brain edema, due largely to astrocyte swelling, and the subsequent increase in intracranial pressure and brain herniation, are major complications of acute liver failure (ALF). Elevated level of brain ammonia has been strongly implicated in the development of astrocyte swelling associated with ALF. The means by which ammonia brings about astrocyte swelling, however, is incompletely understood. Recently, oxidative/nitrosative stress and associated signaling events, including activation of mitogen-activated protein kinases (MAPKs), as well as activation of the transcription factor, nuclear factor-kappaB (NF-κB), have been implicated in the mechanism of ammonia-induced astrocyte swelling. Since these signaling events are known to be regulated by the transcription factor, signal transducer and activator of transcription 3 (STAT3), we examined the state of STAT3 activation in ammonia-treated cultured astrocytes, and determined whether altered STAT3 activation and/or protein expression contribute to the ammonia-induced astrocyte swelling. STAT3 was found to be dephosphorylated (inactivated) at Tyrosine705 in ammonia-treated cultured astrocytes. Total STAT3 protein level was also reduced in ammonia-treated astrocytes. We also found a significant increase in protein tyrosine phosphatase receptor type-1 (PTPRT-1) protein expression in ammonia-treated cultured astrocytes, and that inhibition of PTPRT-1 enhanced the phosphorylation of STAT3 after ammonia treatment. Additionally, exposure of cultured astrocytes to inhibitors of protein tyrosine phosphatases diminished the ammonia-induced cell swelling, while cultured astrocytes over-expressing STAT3 showed a reduction in the astrocyte swelling induced by ammonia. Collectively, these studies strongly suggest that inactivation of STAT3 represents a critical event in the mechanism of the astrocyte swelling associated with acute liver failure.

Abstract 5589: An aqueous extract of Allspice (berries of Pimenta dioica) inhibits breast cancer growth through autophagy by targeting the estrogen receptor

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Breast cancer (BrCa) ranks second as cause of cancer deaths in women. However, >90% of BrCa patients survive >5 years after diagnosis, thus providing an chance to prevent clinical progression and metastasis. Several chemopreventive agents are currently under investigation, however, no single agent is highly effective. Dietary agents offer an advantage for long-term, well tolerated and personalized treatment for chemoprevention. With this goal, we investigated anticancer effects of an aqueous extract of a common spice, the unripe berries of Pimenta dioica (Allspice). Methods: An aqueous exact of Allspice was prepared, lyophilized and a solution of suitable strength was tested on several BrCa cell lines (MCF-7, MDA-MB231, SKBr3) for potential inhibition of clonogenic growth, autophagy and estrogen-receptor inactivation using commonly used analytical techniques. Potential effect on estrogen receptor (ERα) levels and transcriptional activities were determined using quantitative-real time PCR and western blotting. Results: AAE reduced the viability and clonogenic growth of BrCa cells (IC50≈ 100μg/ml). Importantly, AAE was selectively effective against proliferating cells but not quiescent cells. In addition to cytotoxicity, AAE reduced ERα in an estrogen-dependent BrCa cell line, MCF-7. ERα protein levels were significantly decreased in AAE treated MCF7 cells (IC50≈ 100μg/ml). The decrease in ERα protein level by AAE was not rescued by MG132, a proteasome inhibitor, treatment indicating, it is likely due to AAE effect on ERα synthesis and not turnover. We did not detect AAE induced apoptosis in MCF-7 cells as confirmed by several assays. However AAE induced the expressions of autophagy-related proteins, microtubule-associated protein 1 light chain 3 (LC3). Conclusion: We demonstrate anti-proliferative and anti-ERα activity of the aqueous extract of Allspice, a highly aromatic and delectable culinary additive world over. The cytotoxicity of this anticancer agent encompassed multiple pathways, including autophagy and ERα down modulation. These results, if found corroborative using studies in vivo models, should provide a strong rationale to test as an anti-cancer agent in breast cancer. [Grant Support: 1R01AT003544 (BLL)] Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5589. doi:10.1158/1538-7445.AM2011-5589

Abstract 5572: An aqueous extract of allspice (Pimenta dioica) suppresses androgen receptor expression and prostate cancer growth

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background and objective: The aromatic berries of the Jamaican plant Pimenta dioica called Allspice are used in many cuisines for their delectable flavor and as folk remedies for variety of ailments. Despite a rich source of antioxidants the use of Allspice as cancer preventive or anticancer agent has not been reported. Potential anti-cancer activity of an Aqueous Allspice extract (AAE) was tested on prostate cancer (CaP) cells and on tumor models. Methods: AAE was prepared from dried, unripe berries of P. dioica (Certified Organic, Oregon Spices, OR) in water and the extract was lyophilized before testing for biological activity. We tested AAE on growth, survival; response to androgen on human androgen sensitive (AR+) CaP cells (LNCaP, LAPC-4) and in LNCaP xenograft tumors in athymic-nude mice. We used colorimetric MTT-cell survival assay, colony-forming assays, bioluminescence reporter assays, Q-PCR and western blotting to delineate the molecular mechanism of tumor suppressive action of AAE. Results: AAE inhibited tumor cell proliferation (IC50∼100ug/ml) and clonogenic growth (IC50∼40ug/ml) of AR+ CaP cells. AAE was selectively toxic to tumor cells and less toxic to non-tumorigenic cells. AAE blocked cell cycle progression from G1 to S by decreasing cyclin D1, phosphorylated Rb, CDK4 and increased P21 and P27 in AR+ CaP cells. AAE induced apoptosis in LNCaP cells was mediated by intrinsic apoptotic pathway via depolarization of mitochondria, activation of caspase 9, caspase 3/7, cleaved PARP levels, increase in pro-apoptotic protein Bax, decrease in anti-apoptotic protein BID and release of cytochrome C from mitochondria. Importantly, AAE treated AR+ CaP cells showed significant decrease in AR protein level (IC50 50µg/ml) and the proteosome inhibitor MG132 did not rescue the AR protein level decrease by AAE. Transcriptional analysis of androgen receptor (AR) in AR+ CaP cells showed rapid decrease in AR and PSA expression by AAE. Moreover, treatment of LNCaP tumor bearing mice with AAE (100mg/kg, IP, 2x/w) significantly delayed the tumor growth (58%), prolonged the survival period with no systemic toxicity. Ex vivo analyses of LNCaP tumor tissue showed increased apoptosis (70%) in AAE treated mice compared to tissue from vehicle control, with 44% decrease in IGF-1 levels in serum of treated animals. A chemical analysis showed that the major component present on AAE was polyphenol (37% w/v) and devoid of alkaloids and other bioactive compounds. Conclusion: We identified an anticancer product from a New World plant commonly used in cuisines world-over. This extract suppressed the androgen receptor activity in AR+ CaP cells. These studies demonstrate potential use of AAE as a chemotherapeutic or chemopreventive agent against prostate cancer (Funds: NIH1R01 AT 003544 and VA MERIT Grant # 5312.01) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5572. doi:10.1158/1538-7445.AM2011-5572