The Role of Kif4A in Doxorubicin-Induced Apoptosis in Breast Cancer Cells

This study was to investigate the mechanism and role of Kif4A in doxorubicin-induced apoptosis in breast cancer. Using two human breast cancer cell lines MCF-7 (with wild-type p53) and MDA-MB-231 (with mutant p53), we quantitated the expression levels of kinesin super-family protein 4A (Kif4A) and poly (ADP-ribose) Polymerase-1 (PARP-1) by Western blot after doxorubicin treatment and examined the apoptosis by flow cytometry after treatment with doxorubicin and PARP-1 inhibitor, 3-Aminobenzamide (3-ABA). Our results showed that doxorubicin treatment could induce the apoptosis of MCF-7 and MDA-MB-231 cells, the down-regulation of Kif4A and upregulation of poly(ADP-ribose) (PAR). The activity of PARP-1 or PARP-1 activation was significantly elevated by doxorubicin treatment in dose- and time-dependent manners (P < 0.05), while doxorubicin treatment only slightly elevated the level of cleaved fragments of PARP-1 (P > 0.05). We further demonstrated that overexpression of Kif4A could reduce the level of PAR and significantly increase apoptosis. The effect of doxorubicin on apoptosis was more profound in MCF-7 cells compared with MDA-MB-231 cells (P < 0.05). Taken together, our results suggest that the novel role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells is achieved by inhibiting the activity of PARP-1.     

Cdc2 and Cdk2 play critical roles in low dose doxorubicin-induced cell death through mitotic catastrophe but not in high dose doxorubicin-induced apoptosis

In Huh-7 hepatoma cells, low dose (LD) doxorubicin treatment induces cell death through mitotic catastrophe accompanying the formation of large cells with multiple micronuclei, whereas high dose (HD) doxorubicin induces apoptosis. In this study, we investigated the role of Cdc2 and Cdk2 kinase in the regulation of the two modes of cell death induced by doxorubicin. During HD doxorubicin-induced apoptosis, the histone H1-associated activities of Cdc2 and Cdk2 both progressively declined in parallel with reductions in cyclin A and cyclin B protein levels. In contrast, during LD doxorubicin-induced cell death through mitotic catastrophe, the Cdc2 and Cdk2 kinases were transiently activated 1 day post-treatment, with similar changes seen in the protein levels of cyclin A, cyclin B, and Cdc2. Treatment with roscovitine, a specific inhibitor of Cdc2 and Cdk2, significantly blocked LD doxorubicin-induced mitotic catastrophe and cell death, but did not affect HD doxorubicin-induced apoptosis in Huh-7, SNU-398, and SNU-449 hepatoma cell lines. Our results demonstrate that differential regulation of Cdc2 and Cdk2 activity by different doses of doxorubicin may contribute to the induction of two distinct modes of cell death in hepatoma cells, either apoptosis or cell death through mitotic catastrophe.     

Protectors against doxorubicin-induced cardiotoxicity: Flavonoids

Doxorubicin is a widely used anthracycline anticancer agent. Its use may cause cardiomyopathy: in fact, the development of cumulative dose-related cardiotoxicity forms the major limitation of clinical doxorubicin use. We therefore searched for protective agents that combine iron-chelating and oxygen radical-scavenging properties. Moreover, any novel protector should not interfere with the cytostatic activity of doxorubicin. After extensive in vitro screening we found that flavonoids could serve this purpose. In particular 7-monohydroxyethylrutoside almost completely protected against the negative inotropic action of doxorubicin in the electrically paced mouse left atrium model. In vivo it gave full protection at 500 mg/kg intraperitoneally against the doxorubicin-induced ST-interval lengthening in the ECG. Moreover, this protector did not influence the antitumor effect of doxorubicin either in vitro using the human ovarian cell lines A2780 and OVCAR-3 and the human breast cancer cell line MCF-7 or in vivo in A2780 and OVCAR-3 subcutaneous xenografts in nude mice. Comparison of various iron chelators suggest that iron, in contrast to the general assumption, might not play a crucial role in the oxidative stress-induced toxicity of doxorubicin. Moreover, incubation of vascular endothelial cells with doxorubicin produced overexpression of adhesion molecules, which could be inhibited by 7-monohydroxyethylrutoside. From a study in human volunteers, we conclude that an intravenous dose of 1500 mg/m² of 7-monohydroxyethylrutoside is feasible and is safe to be investigated as protection against doxorubicin-induced cardiotoxicity.     

ALDH-2 deficiency increases cardiovascular oxidative stress--evidence for indirect antioxidative properties

Mitochondrial aldehyde dehydrogenase (ALDH-2) reduces reactive oxygen species (ROS) formation related to toxic aldehydes; additionally, it provides a bioactivating pathway for nitroglycerin. Since acetaldehyde, nitroglycerin, and doxorubicin treatment provoke mitochondrial oxidative stress, we used ALDH-2^−/− mice and purified recombinant human ALDH-2 to test the hypothesis that ALDH-2 has an indirect antioxidant function in mitochondria. Antioxidant capacity of purified ALDH-2 was comparable to equimolar doses of glutathione, cysteine, and dithiothreitol; mitochondrial oxidative stress was comparable in C57Bl6 and ALDH-2^−/− mice after acute challenges with nitroglycerin or doxorubicin, whereas chronic acetaldehyde, nitroglycerin, and doxorubicin treatment dose-dependently increased mitochondrial ROS formation and impaired endothelial function to a greater extent in ALDH-2^−/− mice. Maximal nitroglycerin dose applied in vivo lead to a “super-desensitized” nitroglycerin response in isolated ALDH-2^−/− aortas, inaccessible in C57Bl6 mice. Our results suggest that ALDH-2 has an indirect antioxidative property independent of its thiol-moiety in disease states of cardiovascular oxidative stress.     

ABCA1-dependent mobilization of lysosomal cholesterol requires functional Niemann-Pick C2 but not Niemann-Pick C1 protein

Niemann-Pick disease type C (NPC) is caused by mutations leading to loss of function of NPC1 or NPC2 proteins, resulting in accumulation of unesterified cholesterol in late endosomes and lysosomes. We previously reported that expression of the ATP-binding cassette transporter A1 (ABCA1) is impaired in human NPC1^−/− fibroblasts, resulting in reduced HDL particle formation and providing a mechanism for the reduced plasma HDL cholesterol seen in the majority of NPC1 patients. We also found that treatment of NPC1^−/− fibroblasts with an agonist of liver X-receptor corrects ABCA1 expression and HDL formation and reduces lysosomal cholesterol accumulation. We have confirmed that ABCA1 expression is also reduced in NPC2^−/− cells, and found that α-HDL particle formation is impaired in these cells. To determine whether selective up-regulation of ABCA1 can correct lysosomal cholesterol accumulation in NPC disease cells and HDL particle formation, we produced and infected NPC1^−/− and NPC2^−/− fibroblasts with an adenovirus expressing full-length ABCA1 and enhanced green fluorescent protein (AdABCA1-EGFP). ABCA1-EGFP expression in NPC1^−/− fibroblasts resulted in normalization of cholesterol efflux to apolipoprotein A-I (apoA-I) and α-HDL particle formation, plus a marked reduction in filipin staining of unesterified cholesterol in late endosomes/lysosomes. In contrast, AdABCA1-EGFP treatment of NPC2^−/− fibroblasts to normalize ABCA1 expression had no effect on cholesterol efflux to apoA-I or accumulation of excess cholesterol in lysosomes, and only partially corrected α-HDL formation by these cells. These results suggest that correction of ABCA1 expression can bypass the mutation of NPC1 but not NPC2 to mobilize excess cholesterol from late endosomes and lysosomes in NPC disease cells. Expression of ABCA1-EGFP in NPC1^−/− cells increased cholesterol available for esterification and reduced levels of HMG-CoA reductase protein, effects that were abrogated by co-incubation with apoA-I. A model can be generated in which ABCA1 is able to mobilize cholesterol, to join the intracellular regulatory pool or to be effluxed for HDL particle formation, either directly or indirectly from the lysosomal membrane, but not from the lysosomal lumen. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted (LAT1^−/−) cell clones, derived from a heterozygous LAT1^+/− clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1^−/− DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1^−/− cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1^−/− DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1^−/− DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1^+/− DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.     

DNA hypomethylation leads to cGAS‐induced autoinflammation in the epidermis

DNA methylation is a fundamental epigenetic modification, important across biological processes. The maintenance methyltransferase DNMT1 is essential for lineage differentiation during development, but its functions in tissue homeostasis are incompletely understood. We show that epidermis‐specific DNMT1 deletion severely disrupts epidermal structure and homeostasis, initiating a massive innate immune response and infiltration of immune cells. Mechanistically, DNA hypomethylation in keratinocytes triggered transposon derepression, mitotic defects, and formation of micronuclei. DNA release into the cytosol of DNMT1‐deficient keratinocytes activated signaling through cGAS and STING, thus triggering inflammation. Our findings show that disruption of a key epigenetic mark directly impacts immune and tissue homeostasis, and potentially impacts our understanding of autoinflammatory diseases and cancer immunotherapy.     

The DNA Methyltransferase DNMT1 and Tyrosine-Protein Kinase KIT Cooperatively Promote Resistance to 5-Aza-2′-deoxycytidine (Decitabine) and Midostaurin (PKC412) in Lung Cancer Cells

Lung cancer cells are sensitive to 5-aza-2′-deoxycytidine (decitabine) or midostaurin (PKC412), because decitabine restores the expression of methylation-silenced tumor suppressor genes, whereas PKC412 inhibits hyperactive kinase signaling, which is essential for cancer cell growth. Here, we demonstrated that resistance to decitabine (decitabine^R) or PKC412 (PKC412^R) eventually results from simultaneously remethylated DNA and reactivated kinase cascades. Indeed, both decitabine^R and PKC412^R displayed the up-regulation of DNA methyltransferase DNMT1 and tyrosine-protein kinase KIT, the enhanced phosphorylation of KIT and its downstream effectors, and the increased global and gene-specific DNA methylation with the down-regulation of tumor suppressor gene epithelial cadherin CDH1. Interestingly, decitabine^R and PKC412^R had higher capability of colony formation and wound healing than parental cells in vitro, which were attributed to the hyperactive DNMT1 or KIT, because inactivation of KIT or DNMT1 reciprocally blocked decitabine^R or PKC412^R cell proliferation. Further, DNMT1 knockdown sensitized PKC412^R cells to PKC412; conversely, KIT depletion synergized with decitabine in eliminating decitabine^R. Importantly, when engrafted into nude mice, decitabine^R and PKC412^R had faster proliferation with stronger tumorigenicity that was caused by the reactivated KIT kinase signaling and further CDH1 silencing. These findings identify functional cross-talk between KIT and DNMT1 in the development of drug resistance, implying the reciprocal targeting of protein kinases and DNA methyltransferases as an essential strategy for durable responses in lung cancer.     

Chloride ions control the G1/S cell-cycle checkpoint by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells

The aim of the present study is to investigate whether the chloride affects cell growth and cell-cycle progression of cancer cells. In human gastric cancer MKN28 cells, the culture in the Cl⁻-replaced medium (replacement of Cl⁻ by NO₃⁻) decreased the intracellular chloride concentration ([Cl⁻]_i) and inhibited cell growth. The inhibition of cell growth was due to cell-cycle arrest at the G₀/G₁ phase caused by diminution of CDK2 and phosphorylated Rb. The culture of cells in the Cl⁻-replaced medium significantly increased expressions of p21 mRNA and protein without any effects on p53. These observations indicate that chloride ions play important roles in cell-cycle progression by regulating the expression of p21 through a p53-independent pathway in human gastric cancer cells, leading to a novel, unique therapeutic strategy for gastric cancer treatment via control of [Cl⁻]_i.     

Brain cancer stem-like cell genesis from p53-deficient mouse astrocytes by oncogenic Ras

Here, we show that H-ras^V12 causes the p53-knockout mouse astrocytes (p53−/− astrocytes) to be transformed into brain cancer stem-like cells. H-ras^V12 triggers the p53−/− astrocytes to express a Nestin and a Cd133, which are expressed in normal and cancer neural stem cells. H-ras^V12 also induces the formation of a single cell-derived neurosphere under neural stem cell culture conditions. Furthermore, H-ras^V12-overexpressing p53−/− astrocytes (p53−/−ast-H-ras^V12) possess an in vitro self-renewal capacity, and are aberrantly differentiated into Tuj1-positve neurons both in vitro and in vivo. Amongst a variety of Ras-mediated canonical signaling pathways, we demonstrated that the MEK/ERK signaling pathway is responsible for neurosphere formation in p53-deficient astrocytes, whereas the PI3K/AKT signaling pathway is involved in oncogenic transformation in these cells. These findings suggest that the activation of Ras signaling pathways promotes the generation of brain cancer stem-like cells from p53-deficient mouse astrocytes by changing cell fate and transforming cell properties.     

Upregulation of ABC transporters contributes to chemoresistance of sphingosine 1-phosphate lyase-deficient fibroblasts

Sphingosine 1-phosphate (S1P) is an extra- and intracellular mediator that regulates cell growth, survival, migration, and adhesion in many cell types. S1P lyase is the enzyme that irreversibly cleaves S1P and thereby constitutes the ultimate step in sphingolipid catabolism. It has been reported previously that embryonic fibroblasts from S1P lyase-deficient mice (Sgpl1^−/−-MEFs) are resistant to chemotherapy-induced apoptosis through upregulation of B cell lymphoma 2 (Bcl-2) and Bcl-2-like 1 (Bcl-xL). Here, we demonstrate that the transporter proteins Abcc1/MRP1, Abcb1/MDR1, Abca1, and spinster-2 are upregulated in Sgpl1^−/−-MEFs. Furthermore, the cells efficiently sequestered the substrates of Abcc1 and Abcb1, fluo-4 and doxorubicin, in subcellular compartments. In line with this, Abcb1 was localized mainly at intracellular vesicular structures. After 16 h of incubation, wild-type MEFs had small apoptotic nuclei containing doxorubicin, whereas the nuclei of Sgpl1^−/−-MEFs appeared unchanged and free of doxorubicin. A combined treatment with the inhibitors of Abcb1 and Abcc1, zosuquidar and MK571, respectively, reversed the compartmentalization of doxorubicin and rendered the cells sensitive to doxorubicin-induced apoptosis. It is concluded that upregulation of multidrug resistance transporters contributes to the chemoresistance of S1P lyase-deficient MEFs.     

HSP25 down-regulation enhanced p53 acetylation by dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis

Heat shock proteins (HSPs) play important roles in cellular stress resistance. Previous reports had already suggested that HSP27 played multiple roles in preventing doxorubicin-induced cardiotoxicity. Although HSP25 might have biological functions similar to its human homolog HSP27, the mechanism of HSP25 is still unclear in doxorubicin-induced cardiomyocyte apoptosis. To investigate HSP25 biological function on doxorubicin-induced apoptosis, flow cytometry was employed to analyze cell apoptosis in over-expressing HSP25 H9c2 cells in presence of doxorubicin. Unexpectedly, the H9c2 cells of over-expressing HSP25 have no protective effect on doxorubicin-induced apoptosis. Moreover, no detectable interactions were detected by coimmunoprecipitation between HSP25 and cytochrome c, and HSP25 over-expression failed in preventing cytochrome c release induced by doxorubicin. However, down-regulation of endogenous HSP25 by a specific small hairpin RNA aggravates apoptosis in H9c2 cells. Subsequent studies found that HSP25, but not HSP90, HSP70, and HSP20, interacted with SIRT1. Knockdown of HSP25 decreased the interaction between SIRT1 and p53, leading to increased p53 acetylation on K379, up-regulated pro-apoptotic Bax protein expression, induced cytochrome c release, and triggered caspase-3 and caspase-9 activation. These findings indicated a novel mechanism by which HSP25 regulated p53 acetylation through dissociation of SIRT1 from p53 in doxorubicin-induced H9c2 cell apoptosis.     

Ischemia dysregulates DNA methyltransferases and p16INK4a methylation in human colorectal cancer cells

Epigenetic modifications are involved in the initiation and progression of cancer. Expression patterns and activity of DNA methyltransferases (DNMTs) are strictly controlled in normal cells; however, regulation of these enzymes is lost in cancer cells due to unknown reasons. Cancer therapies which target DNMTs are promising treatments of hematologic cancers, but they lack effectiveness in solid tumors. Solid tumors exhibit areas of hypoxia and hypoglycaemia due to their irregular and dysfunctional vasculature, and we previously showed that hypoxia reduces global DNA methylation. Colorectal carcinoma (CRC) cells (HCT116 and 379.2; p53^+/+ and p53^-/-, respectively) were subjected to ischemia (hypoxia and hypoglycaemia) in vitro and levels of DNMTs were assessed. We found a significant decrease in mRNA for DNMT1, DNMT3a and DNMT3b, and similar reductions in DNMT1 and DNMT3a protein levels were detected by western blotting. In addition, total activity levels of DNMTs (as measured by an ELISA-based DNMT activity assay) were reduced in cells exposed to hypoxic and hypoglycaemic conditions. Immunofluorescence of HCT116 tumor xenografts demonstrated an inverse relationship between ischemia (as revealed by carbonic anhydrase IX staining) and DNMT1 protein. Bisulfite sequencing of the proximal promoter region of p16^INK4a showed a decrease in 5-methylcytosine following in vitro exposure to ischemia. These studies provide evidence for the downregulation of DNMTs and modulation of methylation patterns by hypoxia and hypoglycaemia in human CRC cells, both in vitro and in vivo. Our findings suggest that ischemia, either intrinsic or induced through the use of anti-angiogenic drugs, may influence epigenetic patterning and hence tumor progression.Key words: DNA methylation, DNA methyltransferases, colorectal carcinoma, ischemia, p53, hypoxia, hypoglycaemia     

Targeting of 5-aza-2′-deoxycytidine residues by chromatin-associated DNMT1 induces proteasomal degradation of the free enzyme

5-Aza-2′-deoxycytidine (5-aza-dC) is a nucleoside analogue with cytotoxic and DNA demethylating effects. Here we show that 5-aza-dC induces the proteasomal degradation of free (non-chromatin bound) DNMT1 through a mechanism which is dependent on DNA synthesis and the targeting of incorporated 5-aza-dC residues by DNMT1 itself. Thus, 5-aza-dC induces Dnmt1 degradation in wild-type mouse ES cells, but not in Dnmt [3a^–/–, 3b^–/–] mouse ES cells which express Dnmt1 but lack DNA methylation (<0.7% of CpG methylated) and contain few hemi-methylated CpG sites, these being the preferred substrates for Dnmt1. We suggest that adducts formed between DNMT1 and 5-aza-dC molecules in DNA induce a ubiquitin-E3 ligase activity which preferentially targets free DNMT1 molecules for degradation by the proteasome. The proteasome inhibitor MG132 prevents DNMT1 degradation and reduces hypomethylation induced by 5-aza-dC.     

Measurement of doxorubicin-induced lipid peroxidation under the conditions that determine cytotoxicity in cultured tumor cells.

We have investigated doxorubicin-induced lipid peroxidation by the measure of malondialdehyde (MDA) formation in rat glioblastoma cells and human breast carcinoma cells in culture. There was a significant production of MDA when the cells were incubated with pharmacologically relevant doxorubicin concentrations, i.e., concentrations that produce a significant cytotoxicity (0.1 micrograms/ml). At equitoxic doses, vincristine provided no lipid peroxidation, indicating that MDA formation is not a consequence of cell death. Doxorubicin-induced lipid peroxidation was maximal 24 h after incubation of the cells with doxorubicin, indicating that a delay was necessary for the free radical-mediated membrane damage induced by doxorubicin. In the presence of alpha-tocopherol in the culture medium, the doxorubicin-induced MDA formation was inhibited. The development of this method will help in defining the role of free radicals and lipid peroxidation in the cytotoxicity of doxorubicin.     

CDK4 inhibition restores G₁-S arrest in MYCN-amplified neuroblastoma cells in the context of doxorubicin-induced DNA damage

Relapse with drug-resistant disease is the main cause of death in MYCN-amplified neuroblastoma patients. MYCN-amplified neuroblastoma cells in vitro are characterized by a failure to arrest at the G?-S checkpoint after irradiation- or drug-induced DNA damage. We show that several MYCN-amplified cell lines harbor additional chromosomal aberrations targeting p53 and/or pRB pathway components, including CDK4/CCND1/MDM2 amplifications, p16INK4A/p14ARF deletions or TP53 mutations. Cells with these additional aberrations undergo significantly lower levels of cell death after doxorubicin treatment compared with MYCN-amplified cells, with no additional mutations in these pathways. In MYCN-amplified cells CDK4 expression is elevated, increasing the competition between CDK4 and CDK2 for binding p21. This results in insufficient p21 to inhibit CDK2, leading to high CDK4 and CDK2 kinase activity upon doxorubicin treatment. CDK4 inhibition by siRNAs, selective small compounds or p19^INK4D overexpression partly restored G?-S arrest, delayed S-phase progression and reduced cell viability upon doxorubicin treatment. Our results suggest a specific function of p19^INK4D, but not p16^INK4A, in sensitizing MYCN-amplified cells with a functional p53 pathway to doxorubicin-induced cell death. In summary, the CDK4/cyclin D-pRB axis is altered in MYCN-amplified cells to evade a G?-S arrest after doxorubicin-induced DNA damage. Additional chromosomal aberrations affecting the p53-p21 and CDK4-pRB axes compound the effects of MYCN on the G? checkpoint and reduce sensitivity to cell death after doxorubicin treatment. CDK4 inhibition partly restores G?-S arrest and sensitizes cells to doxorubicin-mediated cell death in MYCN-amplified cells with an intact p53 pathway.