Ragu Kanagasabai

Forced Expression of Heat Shock Protein 27 (Hsp27) Reverses P-Glycoprotein (ABCB1)-mediated Drug Efflux and MDR1 Gene Expression in Adriamycin-resistant Human Breast Cancer Cells

Mutant p53 accumulation has been shown to induce the multidrug resistance gene (MDR1) and ATP binding cassette (ABC)-based drug efflux in human breast cancer cells. In the present work, we have found that transcriptional activation of the oxidative stress-responsive heat shock factor 1 (HSF-1) and expression of heat shock proteins, including Hsp27, which is normally known to augment proteasomal p53 degradation, are inhibited in Adriamycin (doxorubicin)-resistant MCF-7 cells (MCF-7/adr). Such an endogenous inhibition of HSF-1 and Hsp27 in turn results in p53 mutation with gain of function in its transcriptional activity and accumulation in MCF-7/adr. Also, lack of HSF-1 enhances nuclear factor κB (NF-κB) DNA binding activity together with mutant p53 and induces MDR1 gene and P-glycoprotein (P-gp, ABCB1), resulting in a multidrug-resistant phenotype. Ectopic expression of Hsp27, however, significantly depleted both mutant p53 and NF-κB (p65), reversed the drug resistance by inhibiting MDR1/P-gp expression in MCF-7/adr cells, and induced cell death by increased G2/M population and apoptosis. We conclude from these results that HSF-1 inhibition and depletion of Hsp27 is a trigger, at least in part, for the accumulation of transcriptionally active mutant p53, which can either directly or NF-κB-dependently induce an MDR1/P-gp phenotype in MCF-7 cells. Upon Hsp27 overexpression, this pathway is abrogated, and the acquired multidrug resistance is significantly abolished so that MCF-7/adr cells are sensitized to Dox. Thus, clinical alteration in Hsp27 or NF-κB level will be a potential approach to circumvent drug resistance in breast cancer.

Hsp27 Protects Adenocarcinoma Cells from UV-Induced Apoptosis by Akt and p21-Dependent Pathways of Survival

Transcriptional activation of p53 target genes, due to DNA damage, causes either apoptosis or survival by cell cycle arrest and DNA repair. However, the regulators of the choice between cell death and survival signaling have not been completely elucidated. Here, we report that human adenocarcinoma cells (MCF-7) survive UV-induced DNA damage by heat shock protein 27 (Hsp27)–assisted Akt/p21 phosphorylation/translocation. Protein levels of the p53 target genes, such as p21, Bcl-2, p38MAPK, and Akt, showed a positive correlation to Hsp27 level during 48 hours postirradiation, whereas p53 expression increased initially but started decreasing after 12 hours. Hsp27 prevented the G1-S phase cell cycle arrest, observed after 8 hours of post–UV irradiation, and PARP-1 cleavage was inhibited. Conversely, silencing Hsp27 enhanced G1-S arrest and cell death. Moreover, use of either Hsp27 or Akt small interference RNA reduced p21 phosphorylation and enhanced its retention in nuclei even after 48 hours postirradiation, resulting in enhanced cell death. Our results showed that Hsp27 expression and its direct chaperoning interaction increases Akt stability, and p21 phosphorylation and nuclear-to-cytoplasm translocation, both essential effects for the survival of UV-induced DNA-damaged cells. We conclude that the role of Hsp27 in cancer is not only for enhanced p53 proteolysis per se, rather it is also a critical determinant in p21 phosphorylation and translocation. Mol Cancer Res; 8(10); 1399–412. ©2010 AACR.

The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II

Dovitinib (TKI258/CHIR258) is a multi-kinase inhibitor in phase III development for the treatment of several cancers. Dovitinib is a benzimidazole-quinolinone compound that structurally resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Dovitinib bound to DNA as shown by its ability to increase the DNA melting temperature and by increases in its fluorescence spectrum that occurred upon the addition of DNA. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258-DNA complex showed that dovitinib could reasonably be accommodated in the DNA minor groove. Because DNA binders are often topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited the catalytic decatenation activity of topoisomerase IIα. It also inhibited the DNA-independent ATPase activity of yeast topoisomerase II which suggested that it interacted with the ATP binding site. Using isolated human topoisomerase IIα, dovitinib stabilized the enzyme-cleavage complex and acted as a topoisomerase IIα poison. Dovitinib was also found to be a cellular topoisomerase II poison in human leukemia K562 cells and induced double-strand DNA breaks in K562 cells as evidenced by increased phosphorylation of H2AX. Finally, dovitinib inhibited the topoisomerase I-catalyzed relaxation of plasmid DNA and acted as a cellular topoisomerase I poison. In conclusion, the cell growth inhibitory activity and the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also, in part, from its ability to target topoisomerase I and topoisomerase II.

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase IIα Isoform.

Pixantrone is a new noncardiotoxic aza-anthracenedione anticancer drug structurally related to anthracyclines and anthracenediones, such as doxorubicin and mitoxantrone. Pixantrone is approved in the European Union for the treatment of relapsed or refractory aggressive B cell non-Hodgkin lymphoma. This study was undertaken to investigate both the mechanism(s) of its anticancer activity and its relative lack of cardiotoxicity. Pixantrone targeted DNA topoisomerase IIα as evidenced by its ability to inhibit kinetoplast DNA decatenation; to produce linear double-strand DNA in a pBR322 DNA cleavage assay; to produce DNA double-strand breaks in a cellular phospho-histone γH2AX assay; to form covalent topoisomerase II-DNA complexes in a cellular immunodetection of complex of enzyme-to-DNA assay; and to display cross-resistance in etoposide-resistant K562 cells. Pixantrone produced semiquinone free radicals in an enzymatic reducing system, although not in a cellular system, most likely due to low cellular uptake. Pixantrone was 10- to 12-fold less damaging to neonatal rat myocytes than doxorubicin or mitoxantrone, as measured by lactate dehydrogenase release. Three factors potentially contribute to the reduced cardiotoxicity of pixantrone. First, its lack of binding to iron(III) makes it unable to induce iron-based oxidative stress. Second, its low cellular uptake may limit its ability to produce semiquinone free radicals and redox cycle. Finally, because the β isoform of topoisomerase II predominates in postmitotic cardiomyocytes, and pixantrone is demonstrated in this study to be selective for topoisomerase IIα in stabilizing enzyme–DNA covalent complexes, the attenuated cardiotoxicity of this agent may also be due to its selectivity for targeting topoisomerase IIα over topoisomerase IIβ.

The anticancer thiosemicarbazones Dp44mT and triapine lack inhibitory effects as catalytic inhibitors or poisons of DNA topoisomerase IIα

The thiosemicarbazones Dp44mT (di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone) and triapine have potent antiproliferative activity and have been evaluated as anticancer agents. While these compounds strongly bind iron and copper, their mechanism(s) of action are incompletely understood. A recent report (Rao et al., Cancer Research 69:948-57, 2009) suggested that Dp44mT may, in part, exert its cytotoxicity through poisoning of DNA topoisomerase IIα. In the present report, a variety of assays were used to determine whether Dp44mT and triapine target topoisomerase IIα. Neither of these compounds inhibited topoisomerase IIα decatenation or induced cleavage of pBR322 DNA in the presence of enzyme. In cells, Dp44mT did not stabilize topoisomerase IIα covalent binding to DNA using an immunoblot band depletion assay, an ICE (immunodetection of complexes of enzyme-to-DNA) assay, and a protein-DNA covalent complex forming assay. Dp44mT did not display cross resistance to etoposide resistant K562 cells containing reduced topoisomerase IIα levels. Synchronized Dp44mT-treated CHO cells did not display a G2/M cell cycle block expected of a topoisomerase II inhibitor. A COMPARE analysis of Dp44mT using the NCI 60-cell line data indicated that inhibition of cell growth was poorly correlated with DNA topoisomerase IIα mRNA levels. In summary, we found no support for the conclusion that Dp44mT inhibits cell growth through the targeting of topoisomerase IIα. Since clinical trials of triapine are underway, it will be important to better understand the intracellular targeting and mechanisms of action of the thiosemicarbazones to support forward development of these agents and newer analogs.

Potent Cytotoxic Arylnaphthalene Lignan Lactones from Phyllanthus poilanei

Two new (1 and 2) and four known arylnaphthalene lignan lactones (3–6) were isolated from different plant parts of Phyllanthus poilanei collected in Vietnam, with two further known analogues (7 and 8) being prepared from phyllanthusmin C (4). The structures of the new compounds were determined by interpretation of their spectroscopic data and by chemical methods, and the structure of phyllanthusmin D (1) was confirmed by single-crystal X-ray diffraction analysis. Several of these arylnaphthalene lignan lactones were cytotoxic toward HT-29 human colon cancer cells, with compounds 1 and 7-O-[(2,3,4-tri-O-acetyl)-α-l-arabinopyranosyl)]diphyllin (7) found to be the most potent, exhibiting IC50 values of 170 and 110 nM, respectively. Compound 1 showed activity when tested in an in vivo hollow fiber assay using HT-29 cells implanted in immunodeficient NCr nu/nu mice. Mechanistic studies showed that this compound mediated its cytotoxic effects by inducing tumor cell apoptosis through activation of caspase-3, but it did not inhibit DNA topoisomerase IIα activity.

Heat shock factor-1 knockout induces multidrug resistance gene, MDR1b, and enhances P-glycoprotein (ABCB1)-based drug extrusion in the heart

Heat-shock factor 1 (HSF-1), a transcription factor for heat-shock proteins (HSPs), is known to interfere with the transcriptional activity of many oncogenic factors. In the present work, we have discovered that HSF-1 ablation induced the multidrug resistance gene, MDR1b, in the heart and increased the expression of P-glycoprotein (P-gp, ABCB1), an ATP binding cassette that is usually associated with multidrug-resistant cancer cells. The increase in P-gp enhanced the extrusion of doxorubicin (Dox) to alleviate Dox-induced heart failure and reduce mortality in mice. Dox-induced left ventricular (LV) dysfunction was significantly reduced in HSF-1−/− mice. DNA-binding activity of NF-κB was higher in HSF-1−/− mice. IκB, the NF-κB inhibitor, was depleted due to enhanced IκB kinase (IKK)-α activity. In parallel, MDR1b gene expression and a large increase in P-gp and lowering Dox loading were observed in HSF-1−/− mouse hearts. Moreover, application of the P-gp antagonist, verapamil, increased Dox loading in HSF-1−/− cardiomyocytes, deteriorated cardiac function in HSF-1−/− mice, and decreased survival. MDR1 promoter activity was higher in HSF-1−/− cardiomyocytes, whereas a mutant MDR1 promoter with heat-shock element (HSE) mutation showed increased activity only in HSF-1+/+ cardiomyocytes. However, deletion of HSE and NF-κB binding sites diminished luminescence in both HSF-1+/+ and HSF-1−/− cardiomyocytes, suggesting that HSF-1 inhibits MDR1 activity in the heart. Thus, because high levels of HSF-1 are attributed to poor prognosis of cancer, systemic down-regulation of HSF-1 before chemotherapy is a potential therapeutic approach to ameliorate the chemotherapy-induced cardiotoxicity and enhance cancer prognosis.

Heat Shock Protein 25-Enriched Plasma Transfusion Preconditions the Heart against Doxorubicin-Induced Dilated Cardiomyopathy in Mice

Extracellular heat shock proteins (eHsps) in the circulation have recently been found to activate both apoptotic and protective signaling in the heart. However, the role of eHsps in doxorubicin (Dox)-induced heart failure has not yet been studied. The objective of the present study was to determine how Dox affects circulating eHsp25 in blood plasma and how eHsp25 affects Dox-induced dilated cardiomyopathy. Wild-type mice [HSF-1(+/+)] were pretreated with 100 μl of heterozygous heat shock factor-1 [HSF-1(+/−)] mouse plasma (which contained 4-fold higher eHsp25 compared with wild-type mice), HSF-1(+/+) plasma, or saline, before treatment with Dox (6 mg/kg). After 4 weeks of this treatment protocol, HSF-1(+/−) plasma-pretreated mice showed increased eHsp25 in plasma and improved cardiac function (percentage of fractional shortening 37.3 ± 2.1 versus 26.4 ± 4.0) and better life span (31 ± 2 versus 22 ± 3 days) compared with the HSF-1(+/+) plasma or saline-pretreated mice. Preincubation of isolated adult cardiomyocytes with HSF-1(+/−) plasma or recombinant human Hsp27 (rhHsp27) significantly reduced Dox-induced activation of nuclear factor-κB and cytokine release and delayed cardiomyocyte death. Moreover, when cardiomyocytes were incubated with fluorescence-tagged rhHsp27, a saturation in binding was observed, suggesting that eHsp25 can bind to surface receptors. Competitive assays with a Toll-like receptor 2 (TLR2) antibody reduced the rhHSP27 binding, indicating that Hsp25 interacts with TLR2. In conclusion, transfusion of Hsp25-enriched blood plasma protected the heart from Dox-induced cardiotoxicity. Hsp25 antagonized Dox binding to the TLR2 receptor on cardiomyocytes.

TRIM72 modulates caveolar endocytosis in repair of lung cells.

Alveolar epithelial and endothelial cell injury is a major feature of the acute respiratory distress syndrome, in particular when in conjunction with ventilation therapies. Previously we showed [Kim SC, Kellett T, Wang S, Nishi M, Nagre N, Zhou B, Flodby P, Shilo K, Ghadiali SN, Takeshima H, Hubmayr RD, Zhao X. Am J Physiol Lung Cell Mol Physiol 307: L449-L459, 2014.] that tripartite motif protein 72 (TRIM72) is essential for amending alveolar epithelial cell injury. Here, we posit that TRIM72 improves cellular integrity through its interaction with caveolin 1 (Cav1). Our data show that, in primary type I alveolar epithelial cells, lack of TRIM72 led to significant reduction of Cav1 at the plasma membrane, accompanied by marked attenuation of caveolar endocytosis. Meanwhile, lentivirus-mediated overexpression of TRIM72 selectively increases caveolar endocytosis in rat lung epithelial cells, suggesting a functional association between these two. Further coimmunoprecipitation assays show that deletion of either functional domain of TRIM72, i.e., RING, B-box, coiled-coil, or PRY-SPRY, abolishes the physical interaction between TRIM72 and Cav1, suggesting that all theoretical domains of TRIM72 are required to forge a strong interaction between these two molecules. Moreover, in vivo studies showed that injurious ventilation-induced lung cell death was significantly increased in knockout (KO) TRIM72(KO) and Cav1(KO) lungs compared with wild-type controls and was particularly pronounced in double KO mutants. Apoptosis was accompanied by accentuation of gross lung injury manifestations in the TRIM72(KO) and Cav1(KO) mice. Our data show that TRIM72 directly and indirectly modulates caveolar endocytosis, an essential process involved in repair of lung epithelial cells through removal of plasma membrane wounds. Given TRIM72s role in endomembrane trafficking and cell repair, we consider this molecule an attractive therapeutic target for patients with injured lungs.

Cytotoxic and natural killer cell stimulatory constituents of Phyllanthus songboiensis

A dichapetalin-type triterpenoid and a dibenzylbutyrolactone-type lignan, together with five known lignans, a known aromatic diterpenoid, and a known acylated phytosterol, were isolated from the aerial parts of Phyllanthus songboiensis, collected in Vietnam. Their structures were determined by interpretation of the spectroscopic data, and the inhibitory activity toward HT-29 human colon cancer cells of all isolates was evaluated by a cytotoxicity assay. The known arylnaphthalene lignan, (+)-acutissimalignan A, was highly cytotoxic toward HT-29 cells, with an IC50 value of 19 nM, but this compound was inactive as a DNA topoisomerase IIα (topo IIα) poison. The known phytosterol, (-)-β-sitosterol-3-O-β-D-(6-O-palmitoyl)glucopyranoside, was found to stimulate natural killer (NK) cells at a concentration of 10μM in the presence of interleukin 12 (IL-12).