Metformin Promotes Anti-tumor Biomarkers in Human Endometrial Cancer Cells.

Metformin (MET) is increasingly implicated in reducing the incidence of multiple cancer types in patients with diabetes. However, similar effects of MET in non-diabetic women with endometrial cancer (EC) remain unknown. In a pilot study, obese non-diabetic women diagnosed with type 1, grade 1/2 EC, and consenting to participate were randomly assigned to receive MET or no MET (control (CON)) during the pre-surgical window between diagnosis and hysterectomy. Endometrial tumors obtained at surgery (MET, n = 4; CON, n = 4) were analyzed for proliferation (Ki67), apoptosis (TUNEL), and nuclear expression of ERα, PGR, PTEN, and KLF9 proteins in tumor glandular epithelial (GE) and stromal (ST) cells. The percentages of immunopositive cells for PGR and for KLF9 in GE and for PTEN in ST were higher while those for ERα in GE but not ST were lower, in tumors of MET vs. CON patients. The numbers of Ki67- and TUNEL-positive cells in tumor GE and ST did not differ between groups. In human Ishikawa endometrial cancer cells, MET treatment (60 µM) decreased cell numbers and elicited distinct temporal changes in ESR1, KLF9, PGR, PGR-B, KLF4, DKK1, and other tumor biomarker mRNA levels. In the context of reduced KLF9 expression (by siRNA targeting), MET rapidly amplified PGR, PGR-B, and KLF4 transcript levels. Our findings suggest that MET acts directly in EC cells to modify steroid receptor expression and signaling network and may constitute a preventative strategy against EC in high-risk non-diabetic women.

Cardioprotective action of the aqueous extract of Terminalia arjuna bark against toxicity induced by doxorubicin.

BACKGROUND: The aqueous extract of Terminalia arjuna (TA) bark (TAAqE) has been shown to have a direct inotropic effect on ventricular myocytes. Active constituents of TAAqE contain various flavonoids and proanthocyanidins, some of which are known to have antioxidant activities. Whether TAAqE affords a cardioprotective action against oxidative stress (OS) remains unclear. PURPOSE: Increased OS is one of the major mechanisms underlying cardiotoxicity induced by doxorubicin (DOX), a commonly-used anticancer agent. The aim of the present study was to investigate potential cardioprotective effect of TAAqE against DOX-induced OS and cardiac dysfunction. METHODS: OS and cytotoxicity were induced by 1 µM DOX for 24 h in H9c2 cells, a cardiac tissue-derived cell line, and left ventricular (LV) dysfunction was induced by intrapleural injection of DOX (accumulative 20 mg/kg body weight) to mice. Cellular oxidative levels and morphology were assessed using microscopy and oxidative-sensitive fluorescent dyes with and without co-treatment with TAAqE. LV function was monitored weekly with echocardiography. RESULTS: TAAqE reduced OS and preserved mitochondria and cell growth of H9c2 cells against DOX treatment. TAAqE (in drinking water) attenuated the decreased LV function and altered myocardial structure caused by DOX treatment. CONCLUSION: TAAqE exerts a protective action against cardiotoxicity caused by DOX in part via suppression of OS. Thus, TAAqE is a promising cardiotonic in adjuvant cancer chemotherapy.

Metabolic history impacts mammary tumor epithelial hierarchy and early drug response in mice.

The emerging links between breast cancer and metabolic dysfunctions brought forth by the obesity pandemic predict a disproportionate early disease onset in successive generations. Moreover, sensitivity to chemotherapeutic agents may be influenced by the patient's metabolic status that affects the disease outcome. Maternal metabolic stress as a determinant of drug response in progeny is not well defined. Here, we evaluated mammary tumor response to doxorubicin in female mouse mammary tumor virus-Wnt1 transgenic offspring exposed to a metabolically compromised environment imposed by maternal high-fat diet. Control progeny were from dams consuming diets with regular fat content. Maternal high-fat diet exposure increased tumor incidence and reduced tumor latency but did not affect tumor volume response to doxorubicin, compared with control diet exposure. However, doxorubicin-treated tumors from high-fat-diet-exposed offspring demonstrated higher proliferation status (Ki-67), mammary stem cell-associated gene expression (Notch1, Aldh1) and basal stem cell-like (CD29(hi)CD24(+)) epithelial subpopulation frequencies, than tumors from control diet progeny. Notably, all epithelial subpopulations (CD29(hi)CD24(+), CD29(lo)CD24(+), CD29(hi)CD24(+)Thy1(+)) in tumors from high-fat-diet-exposed offspring were refractory to doxorubicin. Further, sera from high-fat-diet-exposed offspring promoted sphere formation of mouse mammary tumor epithelial cells and of human MCF7 cells. Untargeted metabolomics analyses identified higher levels of kynurenine and 2-hydroxyglutarate in plasma of high-fat diet than control diet offspring. Kynurenine/doxorubicin co-treatment of MCF7 cells enhanced the ability to form mammosphere and decreased apoptosis, relative to doxorubicin-only-treated cells. Maternal metabolic dysfunctions during pregnancy and lactation may be targeted to reduce breast cancer risk and improve early drug response in progeny, and may inform clinical management of disease.

Characterization of functional capacity of adult ventricular myocytes in long-term culture.

BACKGROUND: Functional properties of freshly isolated adult ventricular myocytes (AVMs) or those of AVMs during first few weeks in culture were well described. However, the functional capacity of these AVMs such as regenerative potential remains unknown, in part, due to the short lifespan of AVMs in culture. This study modified culture conditions that extended the lifespan of AVMs, isolated from adult rat hearts, longer than 6 months. METHODS: Temporal changes in the morphology of individual AVMs, cell-cell interaction, formation of myofibers, self-repair capacity after injury, expression of senescence biomarkers, and contractile function of AVMs over 5 weeks (defined as long-term culture) were chronologically characterized and quantified with live-cell video and fluorescence microscopy, and immunocytochemistry. RESULTS: Cell growth in size reached a plateau after 4 weeks in culture concomitantly with continuous increase in structural remodeling in long-term culture. Dynamic remodeling of AVMs promoted self-contact of filopodia and cell-cell contact where these contained abundant myofilaments, connexin 43 proteins, and high density and high integrity of mitochondria. Such high capacity also enabled self-repair of AVMs after injury, cytokinesis, and formation of myofibers. AVMs in long-term culture displayed spontaneous contraction and importantly were responsive to electrical stimulation. Moreover, AVMs expressed senescence-associated ß-galactosidase, p16, and stress-associated atrial natriuretic peptides that resulted likely from cellular modeling. CONCLUSIONS: Prolonged longevity of AVMs in culture with characteristics of high functional capacity of organelle regeneration and contraction makes them invaluable for further longitudinal mechanistic studies in cardiac (patho)physiology (e.g., hypertrophy and aging), single-cell analysis (e.g., function of hetero-phenotypes) and drug discovery.

The aqueous extract, not organic extracts, of Terminalia arjuna bark exerts cardiotonic effect on adult ventricular myocytes.

The bark of Terminalia arjuna (TA) has been used for centuries in ayurvedic medicine as cardiotonics for treatment of cardiac disorders. It became recently available as over-the-counter supplements marketed for maintaining a healthy heart. However, the cellular mechanism of its cardiotonic effect remains undefined. The present study was designed to investigate the physicochemical property and inotropic effect of the aqueous extract of TA bark (TA(AqE)) on adult rat ventricular myocytes in comparison with extracts prepared sequentially with organic solvents (organic extracts). The kinetics of myocyte contraction and caffeine-induced contraction were analyzed to assess the effect of TA(AqE) on sarcoplasmic reticular (SR) function. The inotropic effect of TA(AqE) was also compared with that of known cardiotonics, isoproterenol (ISO) and ouabain (Ouab). We found that TA(AqE) decoctions exerted positive inotropy, accelerated myocyte relaxation and increased caffeine-induced contraction concentration-dependently. In contrast, TA organic extracts caused interruption of excitability and arrhythmias without consistent inotropic action. In conclusion, TA(AqE)-induced cardiotonic action via enhancing SR function, a unique action minimizing the occurrence of arrhythmias, makes TA(AqE) a promising and relatively safe cardiotonic beneficial to the healthy heart and the treatment for chronic heart disease. The cardiotonic effect of TA(AqE) is consistent with the therapeutic property of TA bark used in ayurvedic medicine. The method of administration and/or selective omission of hydrophobic components from bark powder could be crucial to the efficacy and safety of TA bark in cardiac therapy and uses as over-the-counter supplements.

Inhibition of L-type Ca2+ channel current and negative inotropy induced by arachidonic acid in adult rat ventricular myocytes.

We have previously shown an increase in arachidonic acid (AA) release in response to proinflammatory cytokines in adult rat ventricular myocytes (ARVM). AA is known to alter channel activities; however, its effects on cardiac L-type Ca(2+) channel current (I(Ca,L)) and excitation-contraction coupling remain unclear. The present study examined effects of AA on I(Ca,L), using the whole cell patch-clamp technique, and on cell shortening (CS) and the Ca(2+) transient of ARVM. I(Ca,L) was monitored in myocytes held at -70 mV and internally equilibrated and externally perfused with Na(+)- and K(+)-free solutions. Exposure to AA caused a voltage-dependent block of I(Ca,L) concentration dependently (IC(50) 8.5 microM). The AA-induced inhibition of I(Ca,L) is consistent with its hyperpolarizing shift in the voltage-dependent properties and reduction in maximum slope conductance. In the presence of AA, BSA completely blocked the AA-induced suppression of I(Ca,L) and CS. Intracellular load with AA had no effect on the current density but caused a small depolarizing shift in the I(Ca,L) activation curve, suggesting a site-specific action of AA. Moreover, intracellular AA had no effect on the extracellular AA-induced decrease in I(Ca,L). Pretreatment with indomethacin, an inhibitor of cyclooxygenase, or addition of nordihydroguaiaretic acid, an inhibitor of lipoxygenase, had no effect on AA-induced changes in I(Ca,L). Furthermore, AA suppressed CS and Ca(2+) transients of intact ARVM with no significant effect on SR function and myofilament Ca(2+) sensitivity. Therefore, these results suggest that AA inhibits contractile function of ARVM, primarily due to its direct inhibition of I(Ca,L) at an extracellular site.

Proliferation of human breast cancer cells and anti-cancer action of doxorubicin and vinblastine are independent of PKC-alpha.

Protein kinase C (PKC) has been considered for a potential target of anticancer chemotherapy. PKC-alpha has been associated with growth and metastasis of some cancer cells. However, the role of PKC-alpha in human breast cancer cell proliferation and anticancer chemotherapy remains unclear. In this study, we examined whether alterations of PKC-alpha by phorbol esters and PKC inhibitors could affect proliferation of human breast cancer MCF-7 cells and the cytotoxic effect of chemotherapeutic agents. Exposure for 24 h to doxorubicin (DOX) and vinblastine (VIN) caused a concentration-dependent reduction in proliferation of MCF-7 cells. However, these two anticancer drugs altered cellular morphology and growth pattern in distinct manners. Phorbol 12,13-dibutyrate (PDBu, 100 nM), which enhanced activities of PKC-alpha, increased cancer cell proliferation and attenuated VIN (1 microM)-induced cytotoxicity. These effects were not affected in the presence of 10 nM staurosporine. Phorbol myristate acetate (PMA, 100 nM) that completely depleted PKC-alpha also enhanced cancer cell proliferation and attenuated VIN-induced cytotoxicity. Three potent PKC inhibitors, staurosporine (10 nM), chelerythrine (5 microM) and bisindolylmaleimide-I (100 nM), had no significant effect on MCF-7 cell proliferation; staurosporine and chelerythrine, but not bisindolylmaleimide-I, attenuated VIN-induced cytotoxicity. Moreover, neither phorbol esters nor PKC inhibitors had an effect on cytotoxic effects of DOX (1 microM) on MCF-7 cell proliferation. Thus, these data suggest that MCF-7 cell proliferation or the anti-cancer action of DOX and VIN on breast cancer cells is independent of PKC-alpha.

TNF-alpha-induced impairment of mitochondrial integrity and apoptosis mediated by caspase-8 in adult ventricular myocytes.

Tumor necrosis factor (TNF)-alpha has been shown to induce apoptosis in a variety of cell types including cardiac myocytes. Sphingosine/ceramide and nitric oxide have been associated with apoptosis induced by TNF-alpha; however, signaling mechanisms of TNF-alpha-induced apoptosis in cardiac myocytes are not well defined. This study examined whether alterations in mitochondrial integrity are involved in TNF-alpha-induced apoptosis in adult ventricular myocytes (ARVM) and determined the roles of caspase-8 (an upstream mediator of TNF-alpha receptor-associated signaling) in this process. After incubation for 24-48 h in serum-free culture medium, ARVM underwent spontaneous apoptosis, which remained stable and was not affected by Z-IETD-FMK, a selective caspase-8 inhibitor. Meanwhile, exposure to TNF-alpha resulted in an increase in apoptosis that was detectable at 6 h and became significant after 12 h, when compared to time-controls. After 24-h exposure, TNF-alpha increased caspase-8 activities, mitochondrial cytochrome C (Cyt C) release to the cytosol, accompanied by loss of mitochondrial transmembrane potential (delta psi(m)). Inhibition of caspase-8 activation in the presence of Z-IETD-FMK abolished the TNF-alpha-induced increases in mitochondrial Cyt C release, loss of delta psi(m) and apoptosis. Therefore, these results suggest that TNF-alpha-induced increase in apoptosis in ARVM results from caspase-8-dependent impairment of mitochondrial integrity.

Both cGMP and peroxynitrite mediate chronic interleukin-6-induced negative inotropy in adult rat ventricular myocytes.

We previously showed that chronic exposure to interleukin (IL)-6 decreases contractile and sarcoplasmic reticular (SR) function assessed by postrest potentiation (PRP) via a nitric oxide (NO)-dependent mechanism in adult rat ventricular myocytes (ARVM). Cyclic GMP (cGMP) has been associated with NO-associated negative inotropic effects of IL-6 during acute exposure; however, its role in chronic cardiac effects of IL-6 remains unclear. The present study examined the roles of cGMP and peroxynitrite (ONOO-) in chronic IL-6-induced negative inotropy in ARVM. After ARVM were exposed to IL-6 for 2-24 h, intracellular cGMP contents were time dependently increased; this was mimicked by a NO donor and abolished by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase (sGC), or Rp-8-Br-cGMP, an inhibitor of cGMP-dependent protein kinase G (PKG). Meanwhile, the IL-6-induced decrease in PRP at 2 h was blocked by ODQ or Rp-8-Br-cGMP. By contrast, ODQ or Rp-8-Br-cGMP only attenuated the inhibition of PRP induced by IL-6 after 24 h exposure. Furthermore, IL-6 time dependently increased superoxide anion production and ONOO- formation; the latter was abolished by 5,10,15,20-tetrakis-(4-sulphonatophenyl)-porphyrinato iron (III) (FeTPPS), an ONOO- decomposition catalyst. Interestingly, FeTPPS had no effect on the IL-6-elicited decrease in PRP at 2 h, but attenuated it after 24 h exposure. Moreover, inhibition of sGC/cGMP/PKG, but not ONOO- formation, abolished the IL-6-induced inhibition of kinetics of myocyte contraction during 24 h exposure. We conclude that while the sGC/cGMP/PKG pathway was the primary mechanism for chronic IL-6-induced negative inotropy at 2 h, both sGC/cGMP/PKG and ONOO-, at least in part, mediate the IL-6-induced inhibition of SR function after 24 h exposure.

Inhibition of sarcoplasmic reticular function by chronic interleukin-6 exposure via iNOS in adult ventricular myocytes.

Interleukin (IL)-6 has been shown to decrease cardiac contractility via a nitric oxide synthase (NOS)-dependent pathway during acute exposure. We previously reported that IL-6 decreases contractility and increases inducible NOS (iNOS) in adult rat ventricular myocytes (ARVM) after 2 h exposure. The goal of this study was to investigate the cellular mechanism underlying this chronic IL-6-induced negative inotropy and the role of iNOS. Pretreatment for 2 h with 10 ng ml-1 IL-6 decreased the kinetics of cell shortening (CS) and contractile responsiveness to Ca2+o ([Ca2+]o from(0) to 2 mM) in ARVM. We first examined whether IL-6 reduced Ca2+ influx via L-type Ca2+ -channel current (ICa,L). Whole-cell ICa,L in ARVM was measured under conditions similar to those used for CS measurements, and it was found to be unaltered by IL-6. The sarcoplasmic reticular (SR) function was then assessed by examining postrest potentiation (PRP) and caffeine responsiveness of CS. Results showed that treatment with IL-6 for 2 h significantly decreased PRP, which was concomitant with a decrease in the phosphorylation of phospholamban. Following removal of IL-6, PRP and responsiveness to 10 mM caffeine were also reduced. Meanwhile, the IL-6-induced increase in nitric oxide (NO) production after 2 h (but not 1 h) was abolished by NG-monomethyl-l-arginine (l-NMMA) and 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective inhibitor of iNOS). Furthermore, IL-6-elicited suppressions of PRP and responsiveness to caffeine and Ca2+o were abolished by L-NMMA and AMT. Thus, these results suggest that activation of iNOS mediates IL-6-induced inhibition of SR function in ARVM during chronic exposure.

Sex differences in L-type calcium current after chronic ethanol consumption in rats.

Chronic ethanol consumption elicits a progressive cardiac contractile dysfunction, and studies in rats suggest that this alcoholic heart muscle disease is more pronounced in males than females. Cellular changes associated with the ethanol-induced cardiotoxicity remain largely undefined; however, it is possible that L-type Ca(2+) channel current (I(Ca,L)) is affected. Using whole-cell patch-clamp techniques, this study examined I(Ca,L) in adult ventricular myocytes isolated from male and female P-rats that had consumed drinking water (controls) or a 25% ethanol/water mixture for 14 months. The peak amplitude and maximum conductance of I(Ca,L) were 32 and 26% greater, respectively, in cardiomyocytes isolated from ethanol-consuming compared to control male rats. In contrast, no differences in the amplitude or conductance of I(Ca,L) were observed when comparing myocytes isolated from control and ethanol-consuming females. Ethanol treatment had no significant effects on the kinetics I(Ca,L) inactivation or on steady-state activation and inactivation in either gender. In conclusion, male but not female cardiomyocytes respond to chronic ethanol consumption with an increased I(Ca,L) that may represent a compensatory response to the depressed contractility.

Positive inotropic effect of ceramide in adult ventricular myocytes: mechanisms dissociated from its reduction in Ca2+ influx.

Ceramide, a sphingolipid metabolite produced by activation of sphingomyelinase, has been previously shown to reduce L-type Ca2+ channel current (ICa,L) in adult rat ventricular myocytes; however, its effect on contractile function is unknown. In this study, we investigated the effects of ceramide on excitation-contraction coupling in adult ventricular myocytes and on left ventricular (LV) function in isolated hearts. Surprisingly, in patch-clamped myocytes, ceramide increased contraction concomitant with reductions in ICa,L. In intact myocytes, ceramide increased cell shortening (CS) concurrently with enhancing maximum rates of shortening and relaxation and the duration of contraction. Ceramide also increased the amplitudes of postrest potentiated (PRP) contraction. In fura-PE3-loaded myocytes, ceramide increased systolic Ca2+ and the magnitude and maximum rates of the rising and declining phases of Ca2+ transients. Ceramide-elicited decreases in magnitudes of PRP relative to steady-state contraction and the Ca2+ transient suggest an increased fractional Ca2+ release from the sarcoplasmic reticulum (SR). However, ceramide slightly reduced the caffeine-induced Ca2+ transient and had no significant effect on the amplitude of the PRP-elicited Ca2+ transient. Additionally, the ceramide-induced upward shift in the relationship of contraction and the Ca2+ transient and increase in the Ca2+ responsiveness of CS suggest an increase in myofilament Ca2+ sensitivity. In isolated hearts, ceramide increased LV developed pressure and maximum rates of contraction and relaxation at balloon volumes of 30-50 microl. In summary, regardless of decreasing ICa,L, ceramide elicits distinct positive inotropic and lusitropic effects, resulting probably from enhanced SR Ca2+ release and uptake, and increased Ca2+ sensitivity of ventricular myocytes.

JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes.

Interleukin (IL)-6 decreases cardiac contractility via a nitric oxide (NO)-dependent pathway. However, mechanisms underlying IL-6-induced NO production remain unclear. JAK2/STAT3 and ERK1/2 are two well known signaling pathways activated by IL-6 in non-cardiac cells. However, these IL-6-activated pathways have not been identified in adult cardiac myocytes. In this study, we identified activation of these two pathways during IL-6 stimulation and examined their roles in IL-6-induced NO production and decrease in contractility of adult ventricular myocytes. IL-6 increased phosphorylation of STAT3 (at Tyr(705)) and ERK1/2 (at Tyr(204)) within 5 min that peaked at 15-30 min and returned to basal levels at 2 h. Phosphorylation of STAT3 was blocked by genistein, a protein tyrosine kinase inhibitor, and AG490, a JAK2 inhibitor, but not PD98059, an ERK1/2 kinase inhibitor. The phosphorylation of ERK1/2 was blocked by PD98059 and genistein but not AG490. Furthermore, IL-6 enhanced de novo synthesis of iNOS protein, increased NO production, and decreased cardiac contractility after 2 h of incubation. These effects were blocked by genistein and AG490 but not PD98059. We conclude that IL-6 activated independently the JAK2/STAT3 and ERK1/2 pathways, but only JAK2/STAT3 signaling mediated the NO-associated decrease in contractility.

Alterations in Ca2+ cycling by lysoplasmenylcholine in adult rabbit ventricular myocytes.

We previously reported that lysoplasmenylcholine (LPlasC) altered the action potential (AP) and induced afterdepolarizations in rabbit ventricular myocytes. In this study, we investigated how LPlasC alters excitation-contraction coupling using edge-motion detection, fura-PE3 fluorescent indicator, and perforated and whole cell patch-clamp techniques. LPlasC increased contraction, myofilament Ca(2+) sensitivity, systolic and diastolic free Ca(2+) levels, and the magnitude of Ca(2+) transients concomitant with increases in the maximum rates of shortening and relaxation of contraction and the rising and declining phases of Ca(2+) transients. In some cells, LPlasC induced arrhythmias in a pattern consistent with early and delayed aftercontractions. LPlasC also augmented the caffeine-induced Ca(2+) transient with a reduction in the decay rate. Furthermore, LPlasC enhanced L-type Ca(2+) channel current (I(Ca,L)) and outward currents. LPlasC-induced alterations in contraction and I(Ca,L) were paralleled by its effect on the AP. Thus these results suggest that LPlasC elicits distinct, potent positive inotropic, lusitropic, and arrhythmogenic effects, resulting from increases in Ca(2+) influx, Ca(2+) sensitivity, sarcoplasmic reticular (SR) Ca(2+) release and uptake, SR Ca(2+) content, and probably reduction in sarcolemmal Na(+)/Ca(2+) exchange.