Tumor-associated macrophages (TAM) are heterogeneous in nature and comprise antitumor M1-like (M1-TAM) or pro-tumor M2-like (M2-TAM) TAMs. M2-TAMs are a major component of stroma in breast tumors and enhance metastasis by reducing their phagocytic ability and increasing tumor fibrosis. However, the molecular mechanisms that regulate phenotypic plasticity of TAMs are not well known. Here we report a novel tumor suppressor Slit2 in breast cancer by regulating TAMs in the tumor microenvironment. Slit2 reduced the in vivo growth and metastasis of spontaneous and syngeneic mammary tumor and xenograft breast tumor models. Slit2 increased recruitment of M1-TAMs to the tumor and enhanced the ability of M1-TAMs to phagocytose tumor cells in vitro and in vivo. This Slit2-mediated increase in M1-TAM phagocytosis occurred via suppression of IL6. Slit2 was also shown to diminish fibrosis in breast cancer mouse models by increasing the expression of matrix metalloproteinase 13 in M1-TAMs. Analysis of patient samples showed high Slit2 expression strongly associated with better patient survival and inversely correlated with the abundance of CD163+ TAMs. Overall, these studies define the role of Slit2 in inhibiting metastasis by activating M1-TAMs and depleting tumor fibrosis. Furthermore, these findings suggest that Slit2 can be a promising immunotherapeutic agent to redirect TAMs to serve as tumor killers for aggressive and metastatic breast cancers. In addition, Slit2 expression along with CD163+ TAMs could be used as an improved prognostic biomarker in patients with breast cancer. Significance: This study provides evidence that the antitumor effect of Slit2 in breast cancer occurs by activating the phagocytic activity of M1-like tumor-associated macrophages against tumor cells and diminishing fibrosis.

Isoniazid is used either alone or in combination with other drugs for the treatment of tuberculosis. It is also used for the prevention of tuberculosis. Chronic treatment of Isoniazid may cause severe liver damage leading to acute liver failure. The mechanism through which Isoniazid causes liver damage is investigated. Isoniazid treatment generates reactive oxygen species and induces apoptosis in Hep3B cells. It induces antioxidative and apoptotic genes leading to increase in mRNA expression and protein levels in Hep3B cells. Whole genome expression analysis of Hep3B cells treated with Isoniazid has resulted in differential expression of various genes playing prime role in regulation of apoptotic, antioxidative, DNA damage, cell signaling, cell proliferation and differentiation pathways. Isoniazid increased cytosolic Nrf2 protein level while decreased nuclear Nrf2 protein level. It also decreased ERK1 phosphorylation and treatment of Hep3B cells with ERK inhibitor followed by Isoniazid resulting in increased apoptosis in these cells. Two dimensional gel electrophoresis results have also shown differential expression of various protein species including heat shock proteins, proteins playing important role in oxidative stress, DNA damage, apoptosis, cell proliferation and differentiation. Results suggest that Isoniazid induces apoptosis through oxidative stress and also prevents Nrf2 translocation into the nucleus by reducing ERK1 phosphorylation thus preventing cytoprotective effect.

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Resveratrol (RES) is a natural polyphenol having anti-proliferative activity against breast cancer cells. RES in combination with other chemo modulatory agents, minimizes toxicity and increases efficacy of the treatment. Salinomycin (SAL), a monocarboxylic polyether ionophore is known for selectively targeting breast cancer stem cells. Purpose of the present study was to investigate whether RES in combination with SAL exerts synergistic anti-proliferative activity on breast cancer cells. We further evaluated the molecular mechanism behind SAL and RES mediated cell death. Cytotoxicity assay was performed to determine 50% inhibitory concentration (IC50) of SAL and RES in different human breast cancer cells (HBCCs). Drug synergism and combination index (CI) were calculated using CompuSyn software and effects of synergistic combinations (CI < 1) involving lower doses of SAL and RES were selected for further studies. This combination significantly induced apoptosis in HBCCs without affecting non tumorigenic human breast epithelial cells MCF-10A. Co-treatment enhanced apoptosis in MCF-7 cells via reactive oxygen species (ROS) mediated mitochondrial dysfunction. Oxidative stress disrupt redox homeostasis which altered antioxidant enzymes viz. CuZn Superoxide dismutase (SOD), MnSOD and catalase. Additionally, combination altered nuclear morphology, enhanced PARP cleavage and led to caspase activation. SAL and RES also synergistically modulated MAPK pathway. Study suggests that SAL and RES offer a novel combination approach for the treatment of breast cancer.