Functional analysis of PIK3CA gene mutations in human colorectal cancer

Mutations in the PIK3CA gene, which encodes the p110alpha catalytic subunit of phosphatidylinositol 3-kinase (PI3K), have been reported in human cancers, including colorectal cancer. Most of the mutations cluster at hotspots within the helical and kinase domains. Whereas H1047R, one of the hotspot mutants, is reported to have elevated lipid kinase activity, the functional consequences of other mutations have not been examined. In this study, we examined the effects of colon cancer-associated PIK3CA mutations on the lipid kinase activity in vitro, activation of the downstream targets Akt and p70S6K in vivo and NIH 3T3-transforming ability. Of eight mutations examined, all showed increased lipid kinase activity compared with wild-type p110alpha. All the mutants strongly activated Akt and p70S6K compared with wild-type p110alpha as determined by immunoblotting using phospho-specific antibodies. These mutants also induced morphologic changes, loss of contact inhibition, and anchorage-independent growth of NIH 3T3 cells. The hotspot mutations examined in this study, E542K, E545K, and H1047R, all had high enzymatic and transforming activities. These results show that almost all the colon cancer-associated PIK3CA mutations are functionally active so that they are likely to be involved in carcinogenesis.     

Dietary bioflavonoids induce apoptosis in human leukemia cells

Dietary bioflavonoids are secondary metabolites of plants that are known to have a variety of bio-effects, including anti-cancer activity. In this study, we examined the effects of flavonoids on the growth of human leukemia cells and found that certain flavonoids induce apoptosis in a variety of human leukemia cells. The apoptosis induced by bioflavonoids was dose-dependent and was accompanied by a disruption of the mitochondrial transmembrane potential and the activation of caspase. Our data suggests that dietary bioflavonoids may be useful chemotherapeutic reagents for leukemia patients.     

Inhibition of osteopontin expression and function in oral cancer cell lines by antisense oligonucleotides

We examined expression and function of osteopontin (OPN) in oral cancer cell lines using antisense oligonucleotide (AS). Quantitative real-time RT-PCR showed that expression in BSC-OF cells was significantly higher (10-fold) than that in KB cell. AS-study showed that foci of AS-treated BSC-OF cells possessed thin processes and radiated morphologically, although BSC-OF cells showed round foci. Cell growth in AS-group was lower (<80%) than the control. Invasion ability in AS-group became significantly lower (P<0.01). These results suggest that BSC-OF cell is useful for over-expression of OPN, and that OPN contributes to morphology, growth and invasion.     

Sex difference in ischemic acute renal failure in rats: approach by proteomic analysis

It is known that female rats are resistant to ischemic acute renal failure (ARF), compared with male rats. To elucidate sex differences in ischemic ARF, we searched global protein expression in post-ischemic kidneys using proteomic techniques. Ischemic ARF was induced by 45-min ischemia followed by reperfusion. By proteomic analysis, many male- or female-dominant proteins were detected in sham-operated rat kidneys, and significantly increased or decreased proteins were found in post-ischemic kidneys 2 h after reperfusion, at which there were no significant deterioration in renal function of both sexes. We detected 86 proteins showing more than 1.5-fold significant alterations (p<0.01) in both sexes by ischemia/reperfusion (I/R) treatment. Among the altered proteins, we identified a significantly up-regulated protein in male rat kidneys, meprin alpha, a subunit of meprin which had been reported to play a role in the pathophysiology of I/R-induced ARF. In addition, it is known that a potent meprin alpha inhibitor, actinonin, can protect against I/R-induced renal injury when administered to male rats. We therefore compared the effect of actinonin on I/R-induced renal dysfunction between male and female rats. Renal function of both males and females showed significant deterioration when measured at 24 h after the reperfusion, although the degree of renal dysfunction was much less in females than in males. Pre-ischemic treatment with actinonin (30 mg/kg, i.v.) prevented the I/R-induced renal dysfunction in males but not in females. Our results provide information on differences in protein expression at an early phase after the reperfusion between male and female rats. Moreover, the present study suggests that up-regulation of meprin alpha in the post-ischemic kidney is at least partly involved in aggravation of I/R-induced renal injury in male rats. The possibility that meprin alpha is a key component of the sex difference in ischemic ARF, warrants further attention.     

Bisphenol A 3,4-quinone induces the conversion of xanthine dehydrogenase into oxidase in vitro

In the present study, we assessed the influence of bisphenol A (BPA) and bisphenol A 3,4-quinone (BPAQ) on the conversion of xanthine dehydrogenase (XD) into xanthine oxidase (XO) in the rat liver in vitro. BPA up to 100 μmol/L did not affect the XO and XD activities in the partially purified cytosolic fraction from rat liver, whereas BPAQ (2–10 μmol/L) dose-dependently enhanced the XO activity concomitant with a decrease in the XD activity, implying that BPAQ, but not BPA, can convert XD into the reactive oxygen species (ROS) producing the form XO. Furthermore, it was found that BPAQ could increase the generation of ROS and oxidize the guanine moiety of deoxyguanosine in the DNA of primary rat hepatocyte cultures. These results suggest that BPAQ has the potential to convert XD into XO in the liver, which in turn may lead to ROS generation and oxidative DNA damage in this region.