HKH40A downregulates GRP78/BiP expression in cancer cells.

HKH40A, the 8-methoxy analog of WMC79, is a synthetic agent with promising in vitro and in vivo antitumor activity, especially against solid tumors. However, molecular mechanisms underlying its antitumor effects are poorly understood. Here, we report that HKH40A markedly reduces the level of GRP78/BiP protein in cancer cell lines of various origin. In this study, we show that HKH40A not only downregulates transcription of GRP78 but also directly binds to the isolated protein and induces its proteosomal degradation. Knockdown of BiP increased the efficacy of the drug and overexpression of BiP diminished its activity. BiP is generally highly elevated in solid tumors having a pivotal role in cancer cell survival and chemoresistance, and has been suggested as a novel target for therapeutic intervention. We show that reduction of BiP level by HKH40A impairs its function and induces unfolded protein response as evidenced by the activation of IRE1α, ATF6 and PERK. This leads to a series of downstream events, including sustained eIF2α phosphorylation, increased abundance of spliced XBP1 mRNA and protein levels of ATF4 and CHOP. We also demonstrate that HKH40A inhibited tumor formation in an in vivo xenograft tumor model. Collectively, our data show that HKH40A reduces BiP levels and this could have an important role in the activity of HKH40A against cancer cells.

Biochemical characterization of a recombinant Swainsona canescens calcium-dependent protein kinase (ScCPK1).

Calcium-dependent protein kinases (CPKs) constitute a unique family of kinases involved in many physiological responses in plants. Biochemical and kinetic properties of a recombinant Swainsona canescens calcium-dependent protein kinase (ScCPK1) were examined in this study. The optimum pH and temperature for activity were pH 7.5 and 37 °C, respectively. Substrate phosphorylation activity of ScCPK1 was calmodulin (CaM) independent. Yet CaM antagonists, W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide] and calmidazolium inhibited the activity with IC(50) values of 750 nM and 350 µM, respectively. Both serine and threonine residues were found to be phosphorylated in autophosphorylated ScCPK1 and in histone III-S phosphorylated by ScCPK1. The [Ca(2)(+)] for half maximal activity (K(0.5)) was found to be 0.4 µM for ScCPK1 with histone III-S as substrate. Kinetic analysis showed that K(M) of ScCPK1 for histone III-S was 4.8 µM. These data suggest that ScCPK1 is a functional Ser/Thr kinase, regulated by calcium, and may have a role in Ca(2)(+)-mediated signaling in S. canescens.

Copper, Zinc-Superoxide Dismutase from Clinically Isolated Escherichia coli: Cloning, Analysis of sodC and Its Possible Role in Pathogenicity.

Superoxide dismutase has been discovered within the periplasm of several Gram-negative pathogens. We studied the Cu,Zn-SOD enzyme in Escherichia coli isolated from clinical samples (stool samples) collected from patients suffering from diarrhea. Antibiogram studies of the isolates were carried out to determine the sensitive and resistant strains. The metal co-factor present in the enzyme was confirmed by running samples in native gels and inhibiting with 2 mM potassium cyanide. A 519 bp sodC gene was amplified from resistant and sensitive strains of Escherichia coli. Cloning and sequencing of the sodC gene indicated variation in the protein and amino acid sequences of sensitive and resistant isolates. The presence of sodC in highly resistant Escherichia coli isolates from diarrheal patients indicates that sodC may play role in enhancing the pathogenicity by protecting cells from exogenous sources of superoxide, such as the oxidative burst of phagocytes. The presence of SodC could be one of the factors for bacterial virulence.