No abstract

Drug target identification is a critical step toward understanding the mechanism of action of a drug, which can help one improve the drug's current therapeutic regime and expand the drug's therapeutic potential. However, current in vitro affinity-chromatography-based and in vivo activity-based protein profiling approaches generally face difficulties in discriminating specific drug targets from nonspecific ones. Here we describe a novel approach combining isobaric tags for relative and absolute quantitation with clickable activity-based protein profiling to specifically and comprehensively identify the protein targets of andrographolide (Andro), a natural product with known anti-inflammation and anti-cancer effects, in live cancer cells. We identified a spectrum of specific targets of Andro, which furthered our understanding of the mechanism of action of the drug. Our findings, validated through cell migration and invasion assays, showed that Andro has a potential novel application as a tumor metastasis inhibitor. Moreover, we have unveiled the target binding mechanism of Andro with a combination of drug analog synthesis, protein engineering, and mass-spectrometry-based approaches and determined the drugbinding sites of two protein targets, NF-B and actin. Molecular & Cellular Proteomics 13: 10.1074/mcp. M113.029793, 876-886, 2014.As most drugs exert pharmacological effects by interacting with their target proteins, the identification of these target proteins is a critical step in unraveling the mechanisms of drug action. It is also imperative for our understanding of the pharmacodynamics of a known drug, suggesting potentially unrevealed actions and thus refining future clinical applications of the substance. Traditional approaches used to identify protein targets of a drug typically utilize immobilized drug affinity chromatography coupled with mass spectrometry (MS) 1 (1, 2). These methods can be applied to cell lysates, but not in an in vivo setting, because of the requirement of a solid support. In vitro target profiling might not accurately reflect the drug's actions in the in vivo physiological environment. To overcome this limitation, several groups have used activity-based protein profiling (ABPP) combined with bio-orthogonal click chemistry to identify drug targets both in vitro and in vivo (supplemental Fig. S1) (3-15). ABPP probes exert their functions via covalent reactions with the target proteins or photoaffinity-based labeling via the incorporation of photoreactive groups. With the increasing sensitivity of modern MS platforms, low-abundance protein targets can be successfully identified. Although both conventional affinity chromatography and recent ABPP-based methods allow us to detect a set of candidate protein targets for a drug, it remains difficult to 1 The abbreviations used are: MS, mass spectrometry; ABPP, activity-based protein profiling; ICABPP, clickable activity-based protein profiling; iTRAQ, isobaric tags for relative and absolute quantitation; DMSO, dimethyl sulfoxide; Andro, androgr...

Background: Andrographolide (ADR), the main active component of Andrographis paniculata, displays anticancer activity in various cancer cell lines, among which leukemia cell lines exhibit the highest sensitivity to ADR. In particular, ADR was also reported to have reduced drug resistance in multidrug resistant cell lines. However, the mechanism of action (MOA) of ADR’s anticancer and anti-drug-resistance activities remain elusive. Methods: In this study, we used the MV4-11 cell line, a FLT3 positive acute myeloid leukemia (AML) cell line that displays multidrug resistance, as our experimental system. We first evaluated the effect of ADR on MV4-11 cell proliferation. Then, a quantitative proteomics approach was applied to identify differentially expressed proteins in ADR-treated MV4-11 cells. Finally, cellular processes and signal pathways affected by ADR in MV4-11 cell were predicted with proteomic analysis and validated with in vitro assays. Results: ADR inhibits MV4-11 cell proliferation in a dose- and time-dependent manner. With a proteomic approach, we discovered that ADR inhibited fatty acid synthesis, cellular iron uptake and FLT3 signaling pathway in MV4-11 cells. Conclusions: ADR inhibits MV4-11 cell proliferation through inhibition of fatty acid synthesis, iron uptake and protein synthesis. Furthermore, ADR reduces drug resistance by blocking FLT3 signaling.

The antiestrogen, ICI 182780 (ICI) proves to be clinically useful for the treatment of estrogen receptor positive breast tumours. We report the assessment of the in vivo and in vitro effects of ICI on apoptosis of breast epithelial cells. In vivo, administration of rats with ICI for 3 weeks resulted in a reduction in the size of the lobular structures with the rate of mammary epithelial apoptosis equivalent to 10, 35 and 45% on treatment with 1, 1.5 and 2 mg ICI per kg body weight, respectively. Concomitantly, these treatment led to a 2.0-, 2.2- and 2.5-fold increase in Bax. Similar elevations were also observed in Bad levels which increased 1.7-, 2.6- and 2.7-fold respectively in the ICI treatment as compared to controls. This also resulted in a dose dependent decrease in Bcl-2 and Bcl-xL protein expressions. Growth inhibition and induction of apoptosis were also observed in the MCF-7 cells following in vitro treatment with ICI. This is closely associated with [1] the down-regulation of Bcl-2 and Bcl-xL proteins and [2] upregulation of Bax and Bad, whose gene products are known to be involved the regulation of apoptosis in mammalian cells. Stable over-expression of Bcl-2 resulted in protection of MCF-7 cells from apoptosis and growth inhibitory effects of ICI. Conversely, reduction of Bcl-2 by antisense transfection make MCF-7 cells more sensitive to ICI-induced growth inhibition and apoptosis. These findings suggest that modulation of Bax, Bcl-xL, Bcl-2 and Bad proteins by ICI may be, in part, responsible for the anti-proliferative and apoptotic effect of ICI seen clinically and in animal models.

We previously identified a novel pregnancy-induced growth inhibitory gene, OKL38. To develop a rat model for further characterization of OKL38's role in the initiation and progression of breast and ovarian cancer, we now report the cloning and characterization of three novel rat OKL38 cDNAs that are derived through alternative splicing and differential promoter usage. These three transcripts differ in their 5' untranslated regions but share a common open reading frame that encoded for a 52-kDa protein. OKL38 is mapped to chromosome 19, spanning a region of approximately 15 kb, and contains eight exons. Differential expression of these three rat OKL38 transcripts was observed in liver, kidney, ovary, mammary gland, and uterus. In situ hybridization localized the rat OKL38 transcripts to the luminal epithelial cells of the rat mammary gland and to the granulosa cells in the rat ovary. In vivo studies showed that the RtOKL38-2.0 transcript and protein were regulated by human chorionic gonadotropin in the rat mammary gland and ovary. Importantly, overexpression of RtOKL38-enhanced green fluorescence protein fusion protein in Buffalo rat liver cells resulted in growth inhibition and cell death. Our present findings suggest that OKL38 may function as an effector for human chorionic gonadotropin protection against mammary carcinogenesis, and the availability of the three rat OKL38 cDNAs may help to elucidate the possible role of OKL38 in cellular growth, differentiation, and carcinogenesis.