A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells

At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis.The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-gamma but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-beta producing CD4(+)CD25(+) T cells toward IFN-gamma producing T cells.Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth

The molecular interaction of a copper chelate with human P-glycoprotein

One of the major reasons for multidrug resistance (MDR) in cancer is the overexpression of P-glycoprotein (P-gp, ABCB1), a drug efflux pump. A novel copper complex, namely, copper (II) N-(2-hydroxyacetophenone) glycinate (CuNG) previously synthesized and characterized by the authors had been tested in this study. In a cell-based assay system with human MDR1 cDNA overexpressed mouse fibroblast NIH MDR1-G185 cell line, we demonstrated that this metal complex can directly interact with this transporter. As CuNG increased cellular accumulation of doxorubicin in P-gp-expressing cells, we presumed that of CuNG may be potential to reverse P-gp-mediated drug resistance probably by lowering the P-gp expression at the protein as well as mRNA level. Interestingly, our studies on UIC2 (a conformation sensitive monoclonal antibody) binding assay indicated the direct interaction of CuNG with P-gp. However, CuNG did not compete for the substrate binding as photoaffinity labeling of P-gp with a substrate analog [125I] iodoarylazidoprazosin ([125I] IAAP) showed approximately twofold increase in [125I] IAAP binding in presence of CuNG. In vitro study showed that CuNG significantly stimulated P-gp-specific ATPase activity in isolated membrane preparations from NIH MDR1-G185 cells. This result further confirmed the CuNG–P-gp direct interaction. This study also demonstrated that CuNG has a drug interaction site different from verapamil-, vinblastine- and progesterone-binding sites on P-gp. Taken together, this is the first report of molecular interaction of any Schiff’s base metal chelate CuNG with human P-gp. This information may be useful to design more efficacious nontoxic metal-based drugs as MDR-reversing agents

Induction of apoptosis in human colorectal cancer cell line, HCT-116 by a vanadium- Schiff base complex

Vanadium compounds are well known for their therapeutic interventions against several diseases. Various biochemical attributes of vanadium complexes inspired us to evaluate the cancer cell killing efficacy of the vanadium complex, viz., vanadyl N-(2-hydroxyacetophenone) glycinate [VO(NG)2]. Previously we showed that VO(NG)2 is an effective anticancer agent in in vitro and in vivo cancer models and imposed miniscule side effects. Herein we report that VO(NG)2 is significantly cytotoxic to various cancer cell lines. Furthermore, this redox active vanadyl complex altered the redox homeostatsis of many human cancer cell lines significantly. VO(NG)2 actuates programmed cell death in human colorectal carcinoma cells(HCT-116) through mitochondrial outer membrane permeabilization but in caspase independent manner, possibly by altering cellular redox status and by inflicting DNA damage. Thus, the present work is an attempt to provide many evidences regarding the potent and selective chemotherapeutic efficacy of the novel VO(NG)2

Capsaicin-induced activation of p53-SMAR1 auto-regulatory loop down-regulates VEGF in non-small cell lung cancer to restrain angiogenesis.

Lung cancer is the leading cause of cancer-related deaths worldwide. Despite decades of research, the treatment options for lung cancer patients remain inadequate, either to offer a cure or even a substantial survival advantage owing to its intrinsic resistance to chemotherapy. Our results propose the effectiveness of capsaicin in down-regulating VEGF expression in non-small cell lung carcinoma (NSCLC) cells in hypoxic environment. Capsaicin-treatment re-activated p53-SMAR1 positive feed-back loop in these cells to persuade p53-mediated HIF-1α degradation and SMAR1-induced repression of Cox-2 expression that restrained HIF-1α nuclear localization. Such signal-modulations consequently down regulated VEGF expression to thwart endothelial cell migration and network formation, pre-requisites of angiogenesis in tumor micro-environment. The above results advocate the candidature of capsaicin in exclusively targeting angiogenesis by down-regulating VEGF in tumor cells to achieve more efficient and cogent therapy of resistant NSCLC

Capsaicin inhibits the nuclear localization of HIF-1α by down regulating Cox-2 in a p53-dependent manner.

<p>(A) Immunoblot representing nuclear and cytosolic levels of HIF-1α in capsaicin-treated/p53-shRNA-transfected Hy-A549 cells. (B) HIF-1α expression was monitored in capsaicin-treated Hy-A549 cells by confocal microscopy (magnification 60x). (C) Time-dependent expression of Cox-2-mRNA and -protein in capsaicin-treated Hy-A549 cells was determined by Western blot and RT-PCR. (D) Control siRNA-/Cox-2-siRNA-transfected Hy-A549 cells were treated with capsaicin (37.5 µM; 24 h) and nuclear translocation of HIF-1α was assessed by Western blot analysis (<i>left panel</i>). Transfection efficiency was determined by analyzing the expression of Cox-2 (<i>right panel</i>). (E) Control vector-/Cox-2 cDNA transfected Hy-A549 cells were treated with capsaicin (37.5 µM; 24 h) and analyzed for nuclear and cytosolic expression of HIF-1α (<i>left panel</i>). Transfection efficiency of Cox-2 was also verified (<i>right panel</i>). (F) Control-shRNA-/p53-shRNA-transfected Hy-A549 cells were treated with capsaicin (37.5 µM; 24 h) and expression profiles of Cox-2 both at protein and mRNA level were examined. (G) Time-dependent expression profiles of SMAR1-protein and mRNA in capsaicin-treated Hy-A549 cells were determined by Western blot and RT-PCR (<i>left panels</i>). Control-/p53-shRNA transfected Hy-A549 cells were treated with capsaicin and expression levels of SMAR1 were checked (<i>right panel</i>). (H) Control-/SMAR1-shRNA transfected Hy-A549 cells were treated with capsaicin and immune-blotted with p-Ser¹⁵-p53. α-Actin/H1-Histone/GAPDH were used as internal loading control. Values are mean ± SEM of three independent experiments in each case or representative of typical experiment.</p

Schematic diagram representing molecular mechanisms of capsaicin-mediated down-regulation of pro-angiogenic factor, VEGF.

<p>Schematic diagram representing molecular mechanisms of capsaicin-mediated down-regulation of pro-angiogenic factor, VEGF.</p

Capsaicin suppresses VEGF expression by SMAR1-mediated down regulation of Cox-2.

<p>(A) Control-/SMAR1-shRNA (<i>left panel</i>) or control-vector/SMAR1-cDNA (<i>right panel</i>) transfected Hy-A549 cells were treated with capsaicin were assayed for Cox-2 expression by Western blot and RT-PCR. Transfection efficiency was verified by Western blot (<i>bottom panels</i>). (B) Cox-2 promoter activity was checked in Hy-A549. Schematic representation of the Cox-2 promoter showing the probable SMAR1-binding sites predicted by using the MARWIZ software, PCR run with eight different sets of primers designed for each probable MAR binding site spreading over four regions (−141 bp to −421 bp; −631 bp to −1121 bp; −1262 bp to −1331 bp; −1471 bp to −1891 bp). ChIP assay with anti-SMAR1 was performed on MAR-binding regions of Cox-2 promoter. Input and control IgG was used as internal control and negative control. (C) The relative abundance of SMAR1 on Cox-2 promoter was analyzed in control and capsaicin treated Hy-A549 cells at binding sites 6 & 7 after ChIP of Cox-2 promoter with anti-SMAR1(<i>left panel</i>) and represented graphically (<i>right panel</i>). (D) Control-/SMAR1-shRNA-transfected Hy-A549 cells were treated with capsaicin and analyzed for reporter HIF-1α and VEGF gene expression by RT-PCR. α-Actin/GAPDH were used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p

Capsaicin selectively inhibits VEGF secretion to retard Hy-A549 cell-induced HUVEC cell migration.

<p>(A) Representative phase contrast photomicrographs demonstrating HUVEC migration upon incubation with spent media of Brefeldin-A-pretreated, capsaicin (37.5 µM)-treated Hy-A549 cells (<i>left panel</i>). Percent cell migrated in the wound area has been represented graphically (<i>right panel</i>). (B) Immunoblots showing expression profiles of pro-angiogenic factors VEGF, bFGF, EGF, TGF-β, in presence or absence of capsaicin. (C) Secreted VEGF from cell-free supernatant of Hy-A549 was quantified by ELISA assay (<i>left panel</i>). Time-dependent expression profiles of VEGF-mRNA/-protein in capsaicin-treated Hy-A549 cells were determined by Western blot and RT-PCR respectively (<i>middle panel</i>). Capsaicin-treated Hy-A549 cells were examined for time-dependent variation in the expression profiles of VEGF by quantitative real time PCR analysis and represented graphically (<i>right panel</i>). (D) Immuno-fluorescent images (60x magnification) showing time-dependent pattern of VEGF protein (TRITC-fluorescent) in capsaicin-treated Hy-A549 cells were represented along with nuclear staining (DAPI: blue). (E) Representative images of HUVEC migration upon incubation with (i) recombinant VEGF-supplemented control media, or VEGF-supplemented spent media of capsaicin-treated Hy-A549 cells, or with (ii) anti-VEGF-treated Hy-A549 spent media (<i>left panel</i>). Percent cell migrated in the wound area is being represented graphically (<i>right panel</i>). (F) Representative images of capillary-like sprout formation by HUVECs upon incubation with recombinant VEGF-supplemented spent media of capsaicin-treated Hy-A549 cells or with anti-VEGF-treated Hy-A549 spent media. GAPDH/α-Actin was used as internal loading control. Values are mean ±SEM of three independent experiments in each case or representative of typical experiment.</p