Inhibition of aquaporins as a potential adjunct to breast cancer cryotherapy.

Cryoablation is an emerging type of treatment for cancer. The sensitization of tumors using cryosensitizing agents prior to treatment enhances ablation efficiency and may improve clinical outcomes. Water efflux, which is regulated by aquaporin channels, contributes to cancer cell damage achieved through cryoablation. An increase in aquaporin (AQP) 3 is cryoprotective, whereas its inhibition augments cryodamage. The present study aimed to investigate aquaporin (AQP1, AQP3 and AQP5) gene expression and cellular localization in response to cryoinjury. Cultured breast cancer cells (MDA-MB-231 and MCF-7) were exposed to freezing to induce cryoinjury. RNA and protein extracts were then analyzed using reverse transcription-quantitative PCR and western blotting, respectively. Localization of aquaporins was studied using immunocytochemistry. Additionally, cells were transfected with small interfering RNA to silence aquaporin gene expression and cell viability was assessed using the Sulforhodamine B assay. Cryoinjury did not influence gene expression of AQPs, except for a 4-fold increase of AQP1 expression in MDA-MD-231 cells. There were no clear differences in AQP protein expression for either cell lines upon exposure to frozen and non-frozen temperatures, with the exception of fainter AQP5 bands for non-frozen MCF-7 cells. The exposure of cancer cells to freezing temperatures altered the localization of AQP1 and AQP3 proteins in both MCF-7 and MDA-MD-231 cells. The silencing of AQP1, AQP3 and AQP5 exacerbated MDA-MD-231 cell damage associated with freezing compared with control siRNA. This was also observed with AQP3 and AQP5 silencing in MCF-7 cells. Inhibition of aquaporins may potentially enhance cryoinjury. This cryosensitizing process may be used as an adjunct to breast cancer cryotherapy, especially in the border area targeted by cryoablation where freezing temperatures are not cold enough to induce cellular damage.

Effects of curcumin complexes on MDA­MB­231 breast cancer cell proliferation.

Curcumin displays anticancer properties; however, some issues with the drug delivery mode limit its therapeutic use. Although reformulation and derivatization of curcumin have improved its bioavailability, curcumin derivatives may not retain the same anticancer properties as the parent compound. The present study investigated the anticancer properties of two curcumin complexes, the iron­curcumin [Fe(Cur)3] and boron­curcumin [B(Cur)2] complexes, in the MDA­MB­231 breast cancer cell line. The cellular localization of curcumin, B(Cur)2 and Fe(Cur)3 was determined by fluorescence microscopy. Cell proliferation, migration and invasion were also analysed. Furthermore, apoptosis­associated proteins were detected by using a proteome profiler array, and ion channel gene expression was analysed by reverse transcription­quantitative PCR. The results demonstrated that the three compounds were localized in the perinuclear and cytoplasmic regions of the cell, and displayed cytotoxicity with IC50 values of 25, 35 and 8 µM for curcumin, B(Cur)2 and Fe(Cur)3, respectively. In addition, the three compounds inhibited cell invasion, whereas only curcumin and B(Cur)2 inhibited cell migration. Furthermore, cell exposure to curcumin resulted in an increase in the relative expression of the two key proapoptotic proteins, cytochrome c and cleaved caspase­3, as well as the antiapoptotic protein haem oxygenase­1. In addition, curcumin increased the expression levels of the voltage­gated potassium channels Kv2.1 and Kv3.2. Similarly, the expression levels of the chloride channel bestrophin­1 and the calcium channel coding gene calcium voltage­gated channel auxiliary subunit γ4 were increased following exposure to curcumin. Taken together, these results indicated that Fe(Cur)3 and B(Cur)2 may display similar anticancer properties as curcumin, suggesting that chemical complexation may be considered as a strategy for improving the potency of curcumin in the treatment of breast cancer.

Characterization of BRCA1 and BRCA2 genetic variants in a cohort of Bahraini breast cancer patients using next-generation sequencing.

BACKGROUND: Breast cancer is the most common malignancy in women worldwide. About 5%-10% are due to hereditary predisposition. The contribution of BRCA1/2 mutations to familial breast cancer in Bahrain has not been explored. The objective of this study was to investigate the spectrum of BRCA1/2 genetic variants and estimate their frequencies in familial breast cancer. We also aim to test the efficiency of the next-generation sequencing (NGS) as a powerful tool for detecting genetic variation within BRCA1/2 genes. METHODS: Twenty-five unrelated female patients diagnosed with familial breast cancer were screened for BRCA1/2 variants. All targeted coding exons and exon-intron boundaries of BRCA1/2 genes were amplified with 167 pairs of primers by NGS. RESULTS: We have identified two deleterious BRCA1/2 variants in two patients, one in BRCA1 gene (c.4850C>A) and other in BRCA2 gene (c.67+2T>C). In addition to the deleterious variants, we identified 24 distinct missense variants of uncertain significance, 10 of them are seen to confer minor but cumulatively significant risk of breast cancer. CONCLUSION: Our data suggest that BRCA1/2 variants may contribute to the pathogenesis of familial breast cancer in Bahrain. It also shows that NGS is useful tool for screening BRCA1/2 genetic variants of probands and unaffected relatives.

Curcumin⁻Copper Complex Nanoparticles for the Management of Triple-Negative Breast Cancer.

Breast cancer is the most common cancer diagnosed among females worldwide. Although breast cancer survival has largely improved in the past 30 years, it remains highly heterogeneous in its response to treatment. Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of the estrogen receptor (ER), progesterone receptor (PR) and epidermal growth factor receptor-2 (Her2). While TNBC may initially be responsive to chemotherapy, recurrence and subsequent high mortality rates are frequently reported. Studies have shown curcumin and its derivatives to be effective against TNBC cell lines in vitro. To improve its anti-cancer effects, we have synthesized Fe3+⁻curcumin (Fe⁻Cur3) and Cu2+⁻curcumin (CD) complexes and investigated them experimentally. Further, CD was encapsulated into a poly(styrene)-co-maleic acid (SMA) micelle to enhance its stability. We assessed the cytotoxicity of these formulations both in vitro and in vivo. SMA⁻CD demonstrated dose-dependent cytotoxicity and abolished TNBC tumor growth in vivo. The encapsulation of the curcumin⁻copper complex improved its anti-cancer activity without overt adverse effects in a murine model of TNBC. These results provide evidence and insights into the value of nanoformulations in enhancing drug-delivery and increasing the potential therapeutic efficacy of curcumin derivatives.

Synthetic cannabinoids nano-micelles for the management of triple negative breast cancer.

Triple-negative breast cancer (TNBC) is a highly heterogeneous disease with poor prognosis and inadequate therapeutic outcome. This contribution reports the use of a cannabinoid derivative, WIN55,212-2 (WIN) on the growth of TNBC in a 4T1 syngeneic mouse model. To reduce the well-known psychoactive side effects of cannabinoids, we prepared a nanomicellar formulation of WIN (SMA-WIN). In vivo biodistribution, in silico ADME predictions, anticancer activity, and psychoactive effect of WIN and SMA-WIN studies suggest that SMA-WIN formulation can reduce to greater extent tumor growth with milder psychoactive side effects when compared to free drug. Finally, the effects of WIN and SMA-WIN in combination with doxorubicin (Doxo), an established chemotherapeutic agent for the treatment of TNBC, were investigated in vitro and in vivo. SMA-WIN in combination with Doxo showed therapeutic efficacy and was able to reduce the tumor volume of TNBC murine model drastically. Moreover, SMA-WIN, while favoring drug tumor accumulation, minimized the adverse psychoactive effects that have impeded the use of this agent in the clinic. To our knowledge, this is the first report for the assessment of cannabinoid nanoparticles in vivo for the treatment of TNBC and its enhanced anticancer effect at low doses with Doxo. These findings suggest a new therapeutic strategy in the management of TNBC.

Styrene maleic acid encapsulated raloxifene micelles for management of inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) comprises a group of disorders that manifest through chronic inflammation of the colon and small intestine. Although the exact cause of IBD is still unclear, dysfunctional immunoregulation involving overproduction of inflammatory cytokines such as TNF-α, and IL-6 have been implicated in pathogenesis. Current therapy relies on immunosuppression, cytotoxic drugs, and monoclonal antibodies against TNF-α. These classes of drugs have severe side-effects, especially when used for long duration. Our previous work with raloxifene, a selective estrogen receptor modulator, has shown that the drug, and to a greater extent its micellar formulation, has a significant suppressive effect on NF-κB, an essential immune-regulator. This finding directed the current work towards testing the anti-inflammatory and immunomodulatory effects of raloxifene using cell lines, as well as testing the potential use of the styrene maleic acid (SMA) micelles loaded with raloxifene (SMA-Ral) against dextran sulfate sodium (DSS) induced colitis in an in vivo model of IBD. RESULTS: Treatment of MCF-7 cells with TNF-α was shown to protect the cells from the cytotoxic effect of raloxifene (42 vs. 10% cell death, with TNF-α. Treating CaCo-2 cells with both free and SMA-Ral improved cell survival after exposure to 2% DDS with significantly higher protection with SMA-Ral. Treatment of U-937 with SMA-Ral and free-Ral resulted in down-regulation of TNF-α, IL-1ß, IL-6, and MIP1α, with greater inhibition of the SMA-Ral, compared to free Ral. Balb/c mice treated with raloxifene and SMA-Ral showed weight gain at 14 days, compared to the control group (122, and 115% respectively). Treatment with raloxifene prevented DSS-induced diarrhea in 6/6 of free raloxifene treated mice and in 5/6 mice treated with SMA-Ral. Control group of DSS-treated mice showed average colon length of 7.4 cm compared to 13 cm in the control group. The average colon length was 12.3 and 11.5 cm for raloxifene and SMA-Ral treated groups, respectively. Furthermore, inflammatory cytokines such as IL-6 and TNF-α were reduced in serum of animals treated with free-Ral and SMA-Ral. CONCLUSIONS: Raloxifene and its micellar formulation warrants further studies to understand their effect on the treatment of colitis. Graphical abstract SMA-Raloxifene preparation and its in vivo and in vitro effect on colitis.

A Central Nervous System-Dependent Intron-Embedded Gene Encodes a Novel Murine Fyn Binding Protein.

The interplay between the nervous and immune systems is gradually being unraveled. We previously reported in the mouse the novel soluble immune system factor ISRAA, whose activation in the spleen is central nervous system-dependent. We also showed that ISRAA plays a role in modulating anti-infection immunity. Herein, we report the genomic description of the israa locus, along with some insights into the structure-function relationship of the protein. Our findings revealed that israa is nested within intron 6 of the mouse zmiz1 gene. Protein sequence analysis revealed a typical SH2 binding motif (Y102TEV), with Fyn being the most likely binding partner. Docking simulation showed a favorable conformation for the ISRAA-Fyn complex, with a specific binding mode for the binding of the YTEV motif to the SH2 domain. Experimental studies showed that in vitro, recombinant ISRAA is phosphorylated by Fyn at tyrosine 102. Cell transfection and pull-down experiments revealed Fyn as a binding partner of ISRAA in the EL4 mouse T-cell line. Indeed, we demonstrated that ISRAA downregulates T-cell activation and the phosphorylation of an activation tyrosine (Y416) of Src-family kinases in mouse splenocytes. Our observations highlight ISRAA as a novel Fyn binding protein that is likely to be involved in a signaling pathway driven by the nervous system.

Induction of dissociated cytokine profiles by ISRAA with selective critical involvement of ERK1/2 in its signaling functions.

The immune system-released activating agent (ISRAA) is an immune mediator activated as a result of a nerve stimulus initiated by immune challenge. We have previously demonstrated that ISRAA and tumor necrosis factor (TNF) receptor 1 (TNFR1) share an interspecies-conserved motif (72% homology) that induces the apoptosis and proliferation of human peripheral blood mononuclear cells (hPBMCs) in a dose-dependent manner. In the present study, cytokine profiles were examined in response to the stimulation of hPBMCs with ISRAA. Furthermore, the signaling pathways induced by ISRAA were mapped. The results revealed high measurable levels of TNF-α, interleukin (IL)-6, IL-8, IL-10 and interferon (IFN)-γ, but not IL-4, IL-17 (IL-17A) or transforming growth factor (TGF)-ß. The analysis of signaling pathways revealed the activation of extracellular-regulated protein kinase (ERK)1/2 as a downstream signal in the mitogen­activated protein kinase (MAPK) pathway during TNF­α and IL-6 production and apoptosis, but not during proliferation following stimulation with ISRAA by triggering the Fas-associated protein with death domain (FADD). STAT3 was found to be unphosphorylated in the ISRAA­stimulated hPBMCs, and STAT3 was ubiquitously expressed in unstimulated cells, suggesting that ISRAA has a protein inhibitor of activated STAT (PIAS)-like activity, by functioning as a negative regulator of the effects of STAT3 on the Janus kinase (JAK)/STAT pathway. The determination of the nature of cytokine responses together with the signaling pathways of cellular activity induced by ISRAA paves the way for the investigation of a potential target of ISRAA and for the development of novel therapeutic approaches for the treatment of immune-regulated disorders.

Differential upregulation of the hypothetical transmembrane protein 66 (TMEM66) in multiple sclerosis patients with potential inflammatory response.

The prevalence of multiple sclerosis (MS) in the Gulf region has markedly increased during the last decade, but the mechanisms of the disease have not been investigated. The present study aimed to understand the molecular processes involved in the disease development of the recently emerged MS in this population using microarray technology to investigate differentially-expressed novel genes in MS patients compared to healthy-matched subjects. The expression of the upregulated genes was confirmed by quantitative polymerase chain reaction (qPCR). Furthermore, gene cloning, protein expression and purification were performed followed by testing of the obtained recombinant protein on biological assays, including cell proliferation and cytokine mRNA detection by reverse transcriptase-qPCR. The results showed that out of ~50,000 genes, the hypothetical transmembrane protein-66 gene (TMEM66) exhibited a 3 times higher expression in MS patients compared to healthy subjects. The TMEM66 gene was cloned and its protein showed marked immunological activity relevant to MS since significant proliferation (P<0.05) and augmented induction of the proinflammatory cytokines, interleukin (IL)-6, interferon-γ, tumor necrosis factor-α, and the chemokines, chemokine ligand 5/chemokine receptor 5, macrophage inflammatory protein 1α (MIP-1α) and MIP-1ß were recorded, but not the anti-inflammatory cytokines, IL-4 or IL-2. In conclusion, TMEM66 may be associated with the molecular events of MS and may be considered as an MS biomarker for future personalized medicine management approaches.

An interspecies conserved motif of the mouse immune system-released activating agent (ISRAA) induces proliferative effects on human cells.

We have recently described an immune system-released activating agent (ISRAA) as a nervous system-induced factor that stimulates immune responses in the mouse spleen. However, the human ISRAA has not yet been identified. In this study, we examined the effects of the mouse ISRAA protein on human peripheral blood mononuclear cells (PBMCs), to observe if the biological activity of this molecule is consistent between the two different species. Mouse ISRAA demonstrated dose-dependent dualistic effects on human cells, as 5 µg exhibited positive apoptosis and 50 pg exhibited significant proliferation (P<0.05). Furthermore, immunosuppressed cells from patients undergoing immunosuppressive therapy demonstrated significant proliferation to 50 pg ISRAA (P<0.05). Studies to compare sequences in different species revealed a preserved motif, exhibiting 72% similarity with the interspecies conserved signal peptide motif of tumor necrosis factor receptor 1 (TNFR1). A mutant ISRAA lacking this motif was produced and tested for its biological effects. The mutant ISRAA demonstrated neither apoptotic nor proliferative effects compared with wild type. Therefore, an interspecies conserved domain of ISRAA constitutes the active site of the molecule, and its effects on immunocompromised cells should be investigated for future therapies in the treatment of immunosuppressive disorders.

The pattern of expression and role of triiodothyronine (T3) receptors and type I 5'-deiodinase in breast carcinomas, benign breast diseases, lactational change, and normal breast epithelium.

AIM: : To study the pattern of expression of triiodothyronine (T3) receptors and type I 5'-deiodinase in various breast pathologies comparing malignant and nonmalignant epithelia that include lactational change. METHODS AND RESULTS: A retrospective study was performed on formalin-fixed, paraffin-embedded archival material from 146 cases of carcinomas, normal breast tissue, breast tissue showing lactational change, and benign breast lesions. Archive tissue blocks were selected and sections were cut for immunohistochemistry to study the expression of thyroid hormone receptor α-1 (THR-α1) in the cytoplasm and nuclei of cells in tissues under study. Thick sections were cut for type I 5'-deiodinase evaluation using reverse transcriptional PCR.THR-α1 showed no nuclear expression in the carcinoma group. Combined nuclear and cytoplasmic expression was seen in 47.6%, 63.4%, 64.3%, and 58.3% in the benign, fibrocystic, fibroadenoma, and lactational change groups, respectively, compared with only 17.4% of cases in the carcinoma group. This suggests deregulation of the thyroid hormone in breast cancer. Theories for the possible role of thyroid hormone in the pathogenesis of breast cancer are discussed.Type I 5'-deiodinase was not shown to be differentially expressed in malignant versus nonmalignant groups. CONCLUSIONS: Our study revealed substantial reduction in the protein expression profile of THRs in malignant versus nonmalignant mammary epithelium suggesting a possible role in breast cancer development. The presence of THRs in mammary epithelium seems to be protective against the development of breast cancer. This could serve as a potential prognostic and therapeutic target for breast cancer.

TNF-alpha and IL-8 in acute stroke and the modulation of these cytokines by antiplatelet agents.

Stroke is associated with elevation of several proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin (IL)-8 that are correlated with central nervous system (CNS) injury. Anti-platelet therapies are important agents in stroke management. The role of antiplatelets as anti-inflammatory agents is not known in acute stroke patients. Furthermore, their effect on induction of potential cytokines as TNF-alpha and IL-8 in those patients is still not clear. Thus, we herein examined the induction of TNF-alpha and IL-8 in acute stroke patients and examined the effects of the antiplatelets drugs aspirin, clopidogrel and dipyridamole, and piracetam in their induction. Cytokines were detected intracellularly in leukocytes from patients who had first acute ischemic stroke and from matched controls by immunocytochemistry. The results showed significant increase of spontaneously produced TNF-alpha and IL-8 in patients compared to control. This induction was significantly inhibited differently by each drug and dual drug agents. The data of this work suggest that antiplatelets agents may have a role in inhibition of stroke mediated proinflammatory cytokine effects, which may initiate a new aspect of the role of antiplatelets in the treatment of acute ischemic stroke.