A monomeric mycobacteriophage immunity repressor utilizes two domains to recognize an asymmetric DNA sequence.

Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.

Topical 2'-Hydroxyflavanone for Cutaneous Melanoma.

2'-hydroxyflavanone (2HF) is a dietary flavonoid with anticancer activity towardsmultiple cancers. Here, we report that topically applied 2HF inhibits the growth of intradermalimplants of melanoma in immunocompetent mice. 2HF induced apoptosis and inhibited the growthof the human SK-MEL-24 as well as murine B16-F0 and B16-F10 melanoma cell lines in vitro.Apoptosis was associated with depletion of caspase-3, caspase-9, and PARP1 in B16-F0 and SKMEL-24 cells. Caspase-9 and MEKK-15 were undetected even in untreated B16-F10 cells. Signalingproteins TNFα, and phospho-PDGFR-ß were depleted in all three cell lines; MEKK-15 was depletedby 2HF in SK-MEL-24 cells. 2HF enhanced sunitinib (an MEK and PDGFR-ß inhibitor) and AZD2461 (a PARP1 inhibitor) cytotoxicity. 2HF also depleted the Ral-regulated, stress-responsive,antiapoptotic endocytic protein RLIP76 (RALBP1), the inhibition of which has previously beenshown to inhibit B16-F0 melanoma growth in vivo. Functional inhibition of RLIP76 was evidentfrom inhibition of epidermal growth factor (EGF) endocytosis by 2HF. We found that topicallyapplied 2HF-Pluronic Lecithin Organogel (PLO) gel inhibited B16-F0 and B16-F10 tumorsimplanted in mice and caused no overt toxicity despite significant systemic absorption. 2HFtreatment reduced phospho-AKT, vimentin, fibronectin, CDK4, cyclinB1, and BCL2, whereas itincreased BIM and phospho-AMPK in excised tumors. Several cancer signals are controlled byendocytosis, a process strongly inhibited by RLIP76 depletion. We conclude that 2HF-PLO gel maybe useful for topical therapy of cutaneous metastases of melanoma and could enhance theantineoplastic effects of sunitinib and PARP1 inhibitors. The mechanism of action of 2HF inmelanoma overlaps with RLI76 inhibitors.

Anticancer activity of 2'-hydroxyflavanone towards lung cancer.

In previous studies, we found that 2'-hydroxyflavonone (2HF), a citrus flavonoid, inhibits the growth of renal cell carcinoma in a VHL-dependent manner. This was associated with the inhibition of glutathione S-transferases (GSTs), the first step enzyme of the mercapturic acid pathway that catalyzes formation of glutathione-electrophile conjugates (GS-E). We studied 2HF in small cell (SCLC) and non-small cell (NSCLC) lung cancer cell lines for sensitivity to 2HF antineoplastic activity and to determine the role of the GS-E transporter Rlip (Ral-interacting protein; RLIP76; RALBP1) in the mechanism of action of 2HF. Our results show that 2HF induced apoptosis in both histological types of lung cancer and inhibited proliferation and growth through suppression of CDK4, CCNB1, PIK3CA, AKT and RPS6KB1 (P70S6K) signaling. Increased E-cadherin and reduced fibronectin and vimentin indicated inhibition of epithelial-mesenchymal transition. Additionally, 2HF inhibited efflux of doxorubicin and increased its accumulation in the cells, but did not add to the transport inhibitory effect of anti-Rlip antibodies alone. Binding of Rlip to 2HF was evident from successful purification of Rlip by 2HF affinity chromatography. Consistent with increased drug accumulation, combined treatment with 1-chloro-2, 4-dinitrobenzene, reduced the GI50 of 2HF by an order of magnitude. Results of in-vivo nude mouse xenograft studies of SCLC and NSCLC, which showed that orally administered 2HF inhibited growth of both histological types of lung cancer, confirmed in-vitro study results. Our result suggest that Rlip inhibition is likely a mechanism of action. Our findings are basis of proposing 2HF as therapeutic or preventative drug for lung cancer.