The role of autophagy in cancer development and response to cancer therapy has been a subject of debate. Here we demonstrate that a series of ruthenium(II) complexes containing a β-carboline alkaloid as ligand can simultaneously induce autophagy and apoptosis in tumor cells. These Ru(II) complexes are nuclear permeable and highly active against a panel of human cancer cell lines, with complex 3 displaying activities greater than those of cisplatin. The antiproliferative potentialities of 1-3 are in accordance with their relative lipophilicities, cell membrane penetration abilities, and in vitro DNA binding affinities. Complexes 1-3 trigger release of reactive oxygen species (ROS) and attenuation of ROS by scavengers reduced the sub-G1 population, suggesting ROS-dependent apoptosis. Inhibition of ROS generation also reduces autophagy, indicating that ROS triggers autophagy. Further studies show that suppression of autophagy using pharmacological inhibitors (3-methyladenine and chloroquine) enhances apoptotic cell death.
Despite extensive studies on CD4þCD25þ regulatory T cells (Tregs), their application in adoptive transfer therapies is still not optimal in immune-competent wild-type (WT) animal models. Therefore, it is compelling to search for more potent Tregs for potential clinical application. Mounting evidence has shown that naturally occurring CD8þCD122þ T cells are also Tregs. However, their suppression in allograft rejection, efficiency in suppression and underlying mechanisms remain unclear. Using a murine allotransplantation model, we reported here that CD8þCD122þ Tregs were actually more potent in suppression of allograft rejection and underwent more rapid homeostatic proliferation than their CD4þCD25þ counterparts. Moreover, they produced more IL-10 and were more potent in suppressing T cell proliferation in vitro. Deficiency in IL-10 in CD4þCD25þ and CD8þCD122þ Tregs resulted in their reduced but equal suppression in vivo and in vitro, suggesting that IL-10 is responsible for more effective suppression by CD8þCD122þ than CD4þCD25þ Tregs. Importantly, transfer of CD8þCD122þ Tregs together with the administration of recombinant IL-15 significantly prolonged allograft survival in WT mice. Thus, for the first time, we demonstrate that naturally arising CD8þCD122þ Tregs not only inhibit allograft rejection but also exert this suppression more potently than their CD4þCD25þ counterparts. This novel finding may have important implications for tolerance induction.
Three novel Ru(II) complexes of the general formula [Ru(N-N)(2)(Norharman)(2)](SO(3)CF(3))(2), where N-N = 2,2'-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), 4,7-diphenyl-1,10-phenanthroline (DIP, 3) and Norharman (9H-pyrido[3,4-b]indole) is a naturally occurring β-carboline alkaloid, have been synthesized and characterized. The molecular structures of 1 and 2 have been determined by X-ray diffraction analysis. The cellular uptake efficiencies, in vitro cytotoxicities and apoptosis-inducing properties of these complexes have been extensively explored. Notably, 1-3 exhibit potent antiproliferative activities against a panel of human cancer cell lines with IC(50) values lower than those of cisplatin. Further studies show that 1-3 can cause cell cycle arrest in the G0/G1 phase and induce apoptosis through mitochondrial dysfunction and reactive oxygen species (ROS) generation. In vitro DNA binding studies have also been conducted to provide information about the possible mechanism of action.
We investigated the antioxidative effect of L-ascorbic acid on lipid peroxidation and on secretion and mRNA expression of IL-1alpha and IL-6 after UVA irradiation (20 J/cm2) in cultured human keratinocytes. Lipid peroxidation was measured by (i) high performance liquid chromatography with UV detection of malondialdehyde (MDA) at 256 nm and (ii) spectrometric measurement of thiobarbituric acid-reactive substances (TBARS). To evaluate UV-induced cytotoxicity, we assessed cell membrane damage by measuring lactate dehydrogenase (LDH) release. UVA-induced lipid peroxidation in cultured human keratinocytes was inhibited by ascorbic acid in a concentration-dependent manner: MDA protein equivalent was reduced by 47% (10(-6)), compared to keratinocytes not exposed to L-ascorbic acid (p < 0.05), and the TBARS showed a concentration-dependent decrease of 49% (10(-6) M) in L-ascorbic acid-supplemented cultures compared to controls (p < 0.05). LDH release was decreased by 45% in L-ascorbic acid-supplemented keratinocyte cultures, indicating protection against cell death (p < 0.05). L-Ascorbic acid was able to downregulate IL-1alpha mRNA expression in both UVA-irradiated and nonirradiated cells; however, IL-6 mRNA expression remained unaffected. The secretion of these cytokines was reduced nearly to normal in the presence of L-ascorbic acid. These findings indicate a major cell-protective effect of L-ascorbic acid on UVA-induced lipid peroxidation and the secretion of pro-inflammatory cytokines by UVA-irradiated human keratinocytes.
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