Elucidating how rice (Oryza sativa) takes up nitrate at the molecular level could help improve the low recovery rate (<50%) of nitrogen fertilizer in rice paddies. As a first step toward that goal, we have cloned a nitrate transporter gene from rice called OsNRT1. OsNRT1 is a new member of a growing transporter family called PTR, which consists not only of nitrate transporters from higher plants that are homologs of the Arabidopsis CHL1 (AtNRT1) protein, but also peptide transporters from a wide variety of genera including animals, plants, fungi, and bacteria. However, despite the fact that OsNRT1 shares a higher degree of sequence identity with the two peptide transporters from plants (approximately 50%) than with the nitrate transporters (approximately 40%) of the PTR family, no peptide transport activity was observed when OsNRT1 was expressed in either Xenopus oocytes or yeast. Furthermore, contrasting the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport activity in Xenopus oocytes, with a K m value of approximately 9 mM. Northern-blot and in situ hybridization analysis indicated that OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair. These data strongly indicate that OsNRT1 encodes a constitutive component of a low-affinity nitrate uptake system for rice.
Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. In this study, we demonstrate that fucoidan reduces tumor size in LLC1-xenograft male C57BL/6 mice. Moreover, we found that LLC1-bearing mice continuously fed fucoidan showed greater antitumor activity than mice with discontinuous feeding. Fucoidan inhibited the in vitro growth of lung cancer cells. Transforming growth factor β (TGFβ) receptors (TGFRs) play important roles in the regulation of proliferation and progression, and high TGFRI expression in lung cancer specimens is associated with a worse prognosis. Herein, using lung cancer cells, we found that fucoidan effectively reduces TGFRI and TGFRII protein levels in vivo and in vitro. Moreover, fucoidan reduces TGFR downstream signaling events, including those in Smad2/3 and non-Smad pathways: Akt, Erk1/2, and FAK phosphorylation. Furthermore, fucoidan suppresses lung cancer cell mobility upon TGFβ stimulation. To elucidate how fucoidan decreases TGFR proteins in lung cancer cells, we found that fucoidan enhances the ubiquitination proteasome pathway (UPP)-mediated degradation of TGFRs in A549 and CL1-5 cells. Mechanistically, fucoidan promotes Smurf2 and Smad7 to conjugate TGFRs, resulting in TGF degradation; however, Smurf2-shRNA abolishes fucoidan-enhanced UPP-mediated TGFR degradation. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan, namely decreasing tumor growth by modulating the TGFR/Smad7/Smurf2-dependent axis, leading to TGFR protein degradation and inhibition of lung cancer cell progression in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent or dietary supplementation for lung cancer, acting via the Smurf2-dependent ubiquitin degradation of TGFβ receptors.
Different parts of medicinal herbs have long been used as traditional Chinese drugs for treating many diseases, whereas materials of similar morphology and chemical fingerprints are often misidentified. Analyses of sequence variations in the nuclear ribosomal DNA (rDNA) internal transcribed spacer (ITS) have become a valid method for authentication of medicinal herbs at the intergenic and interspecific levels. DNA extracted from processed materials is usually severely degraded or contaminated by microorganisms, thus generates no or unexpected PCR products. The goal of this study is to apply the ITS fragments selectively amplified with two designed primer sets for efficient and precise authentication of medicinal herbs. The designed primers led to an accurate PCR product of the specific region in ITS2, which was confirmed with DNA extracted from 55 processed medicinal herbs belonging to 48 families. Moreover, the selectively amplified ITS2 authenticated five sets of easily confusable Chinese herbal materials. The designed primers were proven to be suitable for a broad application in the authentication of herbal materials.
Fucoidan, a sulfated polysaccharide extracted from brown algae, exhibits anti-cancer activity. However, the effects and mechanism of fucoidan-induced apoptosis via endoplasmic reticulum (ER) stress is unclear. In this study, we demonstrated that fucoidan prevents tumorigenesis and reduces tumor size in LLC1-xenograft male C57BL/6 mice. Fucoidan induces an ER stress response by activating the PERK-ATF4-CHOP pathway, resulting in apoptotic cell death in vitro and in vivo. Furthermore, ATF4 knockdown abolishes fucoidan-induced CHOP expression and rescues cell viability. Specifically, fucoidan increases intracellular reactive oxygen species (ROS), which increase ATF4 and CHOP in lung cancer cells. Using the ROS scavenger N-acetyl-l-cysteine (NAC), we found that ROS generation is involved in fucoidan-induced ER stress-mediated apoptosis. Moreover, via Toll-like receptor 4 (TLR4) knockdown, we demonstrated that fucoidan-induced ROS and CHOP expression were attenuated. Our study is the first to identify a novel mechanism for the antitumor activity of fucoidan. We showed that fucoidan inhibits tumor viability by activating the TLR4/ROS/ER stress axis and the downstream PERK-ATF4-CHOP pathway, leading to apoptosis and suppression of lung cancer cell progression. Together, these results indicate that fucoidan is a potential preventive and therapeutic agent for lung cancer that acts via activation of ROS-dependent ER stress pathways.
Ling Zhi-8 (LZ-8), an immunomodulatory protein, is derived from and has been cloned from the medicinal mushroom Ganoderma lucidum (Reishi or Ling Zhi); this protein exhibits immunomodulating and antitumor properties. We investigated the effects of recombinant LZ-8 protein (rLZ-8) on the proliferation of A549 human lung cancer cells. Here, we showed that rLZ-8 inhibits cell growth and that this is correlated with increased G(1) arrest. The treatment of A549 cells with rLZ-8 activated p53 and p21 expression, and both the G(1) arrest and the antigrowth effect were found to be p53 dependent. It was further demonstrated that rLZ-8 inhibited tumor growth in mice transplanted with Lewis lung carcinoma cells. Interestingly, rLZ-8 treatment was found to lead to nucleolar stress (or ribosomal stress) as evidenced by inhibition of precursor ribosomal RNA synthesis and reduced polysome formation in A549 cells. These changes resulted in an increasing binding of ribosomal protein S7 to MDM2 and a decreased interaction between MDM2 and p53. Taking these results together, we have identified a novel rLZ-8 antitumor function that positively modulates p53 via ribosomal stress and inhibits lung cancer cell growth in vitro and in vivo. Our current results suggest that rLZ-8 may have potential as a therapeutic intervention for the treatment of cancers that contain wild-type p53 and high expression of MDM2.
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