Signaling Crosstalk of FHIT,p53, and p38 in etoposide-induced apoptosis in MCF-7 cells

Fragile histidine trail (FHIT) is a tumor suppressor in response to DNA damage which has been deleted in various tumors. However, the signaling mechanisms and interactions of FHIT with regard to apoptotic proteins including p53 and p38 in the DNA damage-induced apoptosis are not well described. In the present study, we used etoposide-induced DNA damage in MCF-7 as a model to address these crosstalks. The time course study showed that the expression of FHIT, p53, and p38MAPK started after 1 hour following etoposide treatment. FHIT overexpression led to increase p53 expression, p38 activation, and augmented apoptosis following etoposide-induced DNA damage compared to wild-type cells. However, FHIT knockdown blocked p53 expression, delayed p38 activation, and completely inhibited etoposide-induced apoptosis. Inhibition of p38 activity prevented induction of p53, FHIT, and apoptosis in this model. Thus, activation of p38 upon etoposide treatment leads to increase in FHIT and p53 expression. In p53 knockdown MCF-7, the FHIT induction was hampered but p38 activation was induced in lower doses of etoposide. In p53 knockdown cells, inhibition of p38 induced FHIT expression and apoptosis. Our data demonstrated that the exposure of MCF-7 cells to etoposide increases apoptosis through a mechanism involving the activation of the p38-FHIT-p53 pathway. Moreover, our findings suggest signaling interaction for these pathways may represent a promising therapy for breast cancer.     

The anti-cancer drug etoposide can induce caspase-8 processing and apoptosis in the absence of CD95 receptor-ligand interaction

Caspase-8 (FLICE) can associate with and be activated by CD95 (APO-1/Fas), an apoptosis-inducing member of the Tumour Necrosis Factor receptor family. We find that, in Jurkat T cells, the DNA damaging anti-cancer drug etoposide induces apoptosis and, surprisingly, processing of caspase-8. Therefore, we have investigated whether etoposide involves CD95 receptor activation. We find that etoposide does not induce CD95 ligand expression at the mRNA level. In addition, blocking of CD95 receptor function with a specific antibody does not inhibit etoposide-induced apoptosis. Apparently, in Jurkat cells, etoposide can induce caspase-8 processing and apoptosis in a CD95-independent fashion. Likewise, we find that thymocytes from the CD95-deficient lpr/lpr mouse strain readily undergo apoptosis in response to etoposide. Moreover, since inhibition of the secretory pathway with brefeldin A does not inhibit etoposide-induced apoptosis, we exclude the requirement for a newly synthesizedreceptor ligand to induce the apoptotic pathway. We conclude that, at least in certain cell types, etoposide does not require CD95 receptor function to induce caspase-8 processing and apoptosis.     

Signaling crosstalk of FHIT,CHK2 and p38 in etoposide induced growth inhibition in MCF-7 cells

FHIT (Fragile Histidin Triad) is a tumor suppressor gene involved in regulating cell death during DNA damage conditions. The exact mechanism of DNA damage-induced FHIT signaling is not well understood. It is known that p38 kinase and CHK2 kinase are being activated during stress-induced conditions and DNA damage, resulting in cell death. Since both CHK2 and FHIT are being influenced by DNA damage, we have evaluated the interplay of p38, CHK2 and FHIT in response to etoposide-induced cell death. DNA damage was induced by etoposide in MCF-7 cells and viability was examined using MTT. FHIT expression was blocked using siRNA. Protein expression was measured using western blotting. Our results indicated that etoposide induced cytotoxicity in MCF-7. Block of FHIT expression, completely reversed etoposide cytotoxicity. Besides, etoposide induced p38 and CHK2 phosphorylation and reduced FHIT expression in a time-dependent manner. The time-course study indicated that CHK2 had been phosphorylated prior to p38 activation. Knockdown of FHIT expression reduced CHK2 phosphorylation but had no significant effect on p38 activation. Inhibition of p38 kinase and CHK2 prevented etoposide induced alteration in FHIT expression. Furthermore, p38 inhibitors augmented etoposide-induced CHK2 phosphorylation. These results indicate that etoposide lowers FHIT expression and induces cell death via p38 and CHK2 phosphorylation. These results demonstrate a time dependent complex crosstalk of FHIT, p38 and CHK2 pathways in response to etoposide. Moreover, our findings suggest signaling interaction for these pathways which can be targeted for manipulating cell proliferation.     

Ascorbate promotes the cellular accumulation of doxorubicin and reverses the multidrug resistance in breast cancer cells by inducing ROS-dependent ATP depletion

Chemotherapy is the most effective strategy for the treatment of metastatic breast cancer. However, P-glycoprotein (P-gp)-mediated multidrug resistance severely limits the efficacy of chemotherapy and is a major cause of the failure during chemotherapeutic treatment. In this study, we investigated the anticancer effects of combining chemotherapeutic drugs with ascorbate (AA) in human breast cancer cells. We found that combined administration of AA can improve the sensitivity of both MCF-7 and doxorubicin (Dox)-resistant MCF-7/Adr cells to Dox in vitro and in vivo by a reactive oxygen species (ROS)-dependent mechanism. Further studies proved that AA can promote the accumulation of Dox in MCF-7/Adr cells when combined with doxorubicin. AA had no effect on the expression of P-gp at the mRNA and protein levels, but could decrease its activity as demonstrated by an obvious inhibition of efflux of P-gp substrate Rh 123. AA reduced ATP levels in both MCF-7 and MCF-7/Adr cells, and pretreating AA-stimulating cells with catalase completely rescued ATP levels. With ATP reduction, we observed an increased cellular calcium and the appearance of vacuoles and micropores on the cell surface, indicating the increased cell membrane permeability in AA-treated MCF-7/Adr cells. The above results suggest that AA could promote the cellular accumulation of doxorubicin by inducing ROS-dependent ATP depletion. Clinically, a combination of AA with doxorubicin would be a novel strategy for reversal of the multidrug resistance in human breast cancer cells during chemotherapy.     

Chronic Oxidative Stress Increases Growth and Tumorigenic Potential of MCF-7 Breast Cancer Cells

Accumulating evidence suggests that exposures to elevated levels of either endogenous estrogen or environmental estrogenic chemicals are associated with breast cancer development and progression. These natural or synthetic estrogens are known to produce reactive oxygen species (ROS) and increased ROS has been implicated in both cellular apoptosis and carcinogenesis. Though there are several studies on direct involvement of ROS in cellular apoptosis using short-term exposure model, there is no experimental evidence to directly implicate chronic exposure to ROS in increased growth and tumorigenicity of breast cancer cells. Therefore, the objective of this study was to evaluate the effects of chronic oxidative stress on growth, survival and tumorigenic potential of MCF-7 breast cancer cells. MCF-7 cells were exposed to exogenous hydrogen peroxide (H₂O₂) as a source of ROS at doses of 25 µM and 250 µM for acute (24 hours) and chronic period (3 months) and their effects on cell growth/survival and tumorigenic potential were evaluated. The results of cell count, MTT and cell cycle analysis showed that while acute exposure inhibits the growth of MCF-7 cells in a dose-dependent manner, the chronic exposure to H₂O₂-induced ROS leads to increased cell growth and survival of MCF-7 cells. This was further confirmed by gene expression analysis of cell cycle and cell survival related genes. Significant increase in number of soft agar colonies, up-regulation of pro-metastatic genes VEGF, WNT1 and CD44, whereas down-regulation of anti-metastatic gene E-Cadherin in H₂O₂ treated MCF-7 cells observed in this study further suggests that persistent exposure to oxidative stress increases tumorigenic and metastatic potential of MCF-7 cells. Since many chemotherapeutic drugs are known to induce their cytotoxicity by increasing ROS levels, the results of this study are also highly significant in understanding the mechanism for adaptation to ROS-induced toxicity leading to acquired chemotherapeutic resistance in breast cancer cells.     

The anti-tumor effect of A3 adenosine receptors is potentiated by pulsed electromagnetic fields in cultured neural cancer cells

A(3) adenosine receptors (ARs) play a pivotal role in the development of cancer and their activation is involved in the inhibition of tumor growth. The effects of pulsed electromagnetic fields (PEMFs) on cancer have been controversially discussed and the detailed mechanisms are not yet fully understood. In the past we have demonstrated that PEMFs increased A(2A) and A(3)AR density and functionality in human neutrophils, human and bovine synoviocytes, and bovine chondrocytes. In the same cells, PEMF exposure increased the anti-inflammatory effect mediated by A(2A) and/or A(3)ARs. The primary aim of the present study was to evaluate if PEMF exposure potentiated the anti-tumor effect of A(3)ARs in PC12 rat adrenal pheochromocytoma and U87MG human glioblastoma cell lines in comparison with rat cortical neurons. Saturation binding assays and mRNA analysis revealed that PEMF exposure up-regulated A(2A) and A(3)ARs that are well coupled to adenylate cyclase activity and cAMP production. The activation of A(2A) and A(3)ARs resulted in the decrease of nuclear factor-kappa B (NF-kB) levels in tumor cells, whilst only A(3)ARs are involved in the increase of p53 expression. A(3)AR stimulation mediated an inhibition of tumor cell proliferation evaluated by thymidine incorporation. An increase of cytotoxicity by lactate dehydrogenase (LDH) release and apoptosis by caspase-3 activation in PC12 and U87MG cells, but not in cortical neurons, was observed following A(3)AR activation. The effect of the A(3)AR agonist in tumor cells was enhanced in the presence of PEMFs and blocked by using a well-known selective antagonist. Together these results demonstrated that PEMF exposure significantly increases the anti-tumor effect modulated by A(3)ARs.     

Numerical Analysis of Etoposide Induced DNA Breaks

Background

Etoposide is a cancer drug that induces strand breaks in cellular DNA by inhibiting topoisomerase II (topoII) religation of cleaved DNA molecules. Although DNA cleavage by topoisomerase II always produces topoisomerase II-linked DNA double-strand breaks (DSBs), the action of etoposide also results in single-strand breaks (SSBs), since religation of the two strands are independently inhibited by etoposide. In addition, recent studies indicate that topoisomerase II-linked DSBs remain undetected unless topoisomerase II is removed to produce free DSBs.

Methodology/Principal Findings

To examine etoposide-induced DNA damage in more detail we compared the relative amount of SSBs and DSBs, survival and H2AX phosphorylation in cells treated with etoposide or calicheamicin, a drug that produces free DSBs and SSBs. With this combination of methods we found that only 3% of the DNA strand breaks induced by etoposide were DSBs. By comparing the level of DSBs, H2AX phosphorylation and toxicity induced by etoposide and calicheamicin, we found that only 10% of etoposide-induced DSBs resulted in histone H2AX phosphorylation and toxicity. There was a close match between toxicity and histone H2AX phosphorylation for calicheamicin and etoposide suggesting that the few etoposide-induced DSBs that activated H2AX phosphorylation were responsible for toxicity.

Conclusions/Significance

These results show that only 0.3% of all strand breaks produced by etoposide activate H2AX phosphorylation and suggests that over 99% of the etoposide induced DNA damage does not contribute to its toxicity.     

Synthesis and biological evaluation of halogenated phenoxychalcones and their corresponding pyrazolines as cytotoxic agents in human breast cancer

Novel halogenated phenoxychalcones 2a–f and their corresponding N-acetylpyrazolines 3a–f were synthesised and evaluated for their anticancer activities against breast cancer cell line (MCF-7) and normal breast cell line (MCF-10a), compared with staurosporine. All compounds showed moderate to good cytotoxic activity when compared to control. Compound 2c was the most active, with IC₅₀ = 1.52 µM and selectivity index = 15.24. Also, chalcone 2f showed significant cytotoxic activity with IC₅₀ = 1.87 µM and selectivity index = 11.03. Compound 2c decreased both total mitogen activated protein kinase (p38α MAPK) and phosphorylated enzyme in MCF-7 cells, suggesting its ability to decrease cell proliferation and survival. It also showed the ability to induce ROS in MCF-7 treated cells. Compound 2c exhibited apoptotic behaviour in MCF-7 cells due to cell accumulation in G2/M phase and elevation in late apoptosis 57.78-fold more than control. Docking studies showed that compounds 2c and 2f interact with p38alpha MAPK active sites.     

Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis

DNA damage induced by the cancer chemotherapeutic drug etoposide triggers the onset of a series of intracellular events characteristic of apoptosis. Among the early changes observed is the release of cytochrome c from mitochondria, although the mechanism responsible for this effect is unclear. We demonstrate here a role for caspase-2 in etoposide-induced cytochrome c release. In particular, Jurkat T-lymphocytes treated with an irreversible caspase-2 inhibitor, benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk), or stably transfected with pro-caspase-2 antisense (Casp-2/AS) are refractory to cytochrome c release stimulated by etoposide. Experiments performed using a reconstituted cell-free system indicate that etoposide-induced cytochrome c release by way of caspase-2 occurs independently of cytosolic factors, suggesting that the nuclear pool of pro-caspase-2 is critical to this process. Apart from inhibiting cytochrome c release, undermining caspase-2 activity results in an attenuation of downstream events, such as pro-caspase-9 and -3 activation, phosphatidylserine exposure on the plasma membrane, and DNA fragmentation. Taken together, our data indicate that caspase-2 provides an important link between etoposide-induced DNA damage and the engagement of the mitochondrial apoptotic pathway.     

(–)-Xanthatin as a Killer of Human Breast Cancer MCF-7 Mammosphere Cells: A Comparative Study with Salinomycin

Experimental evidence accumulated by our research group and others strongly suggests that (–)-xanthatin, a xanthanolide sesquiterpene lactone, exhibits anti-proliferative effects on human breast cancer cells (in vitro) as well as anti-tumor effects in experimental animals (in vivo). In cancer biology, it is now critically important for anti-cancer agents to selectively target cancer stem cells (CSCs) in order to overcome cancer therapeutic resistance and recurrence. However, it has not yet been established whether (–)-xanthatin abrogates the formation of breast CSCs. In the present study, we utilized chemically synthesized pure (–)-xanthatin and a culture system to obtain mammospheres from human breast cancer MCF-7 cells, which are a CSC-enriched population. We herein demonstrated for the first time that (–)-xanthatin exhibited the ability to kill mammospheres, similar to salinomycin, an established selective killer of CSCs. A possible anti-proliferative mechanism toward mammospheres by (–)-xanthatin is discussed.     

Induction of ATM Activation,Histone H2AX Phosphorylation and Apoptosis by Etoposide: Relation to Cell Cycle Phase

Etoposide (VP-16) belongs to the family of DNA topoisomerase II (topo2) inhibitors, drugs widely used in cancer chemotherapy. Their presumed mode of action is stabilization of “cleavable complexes” between topo2 and DNA; collisions of DNA replication forks with these complexes convert them into DNA double-strand breaks (DSBs), potentially lethal lesions that may trigger apoptosis. Immunocytochemical detection of activation of ATM (ATM-S1981P) and histone H2AX phosphorylation (γH2AX) provides a sensitive probe of the induction of DSBs in individual cells. Using multiparameter cytometry we measured the expression of ATM-S1981P and γH2AX as well as initiation of apoptosis (caspase-3 activation) in relation to the cell cycle phase in etoposide-treated human lymphoblastoid TK6 cells. The induction of ATM-S1981P and γH2AX was seen in all phases of the cell cycle. The G1-phase cells, however, preferentially underwent apoptosis. The extent of etoposide-induced H2AX phosphorylation was partially reduced by N-acetyl-L-cysteine (NAC), a scavenger of reactive oxygen species (ROS).The maximal reduction of H2AX phosphorylation by NAC, seen in G1-phase cells, was nearly 50%. NAC also protected a fraction of G1 cells from etoposide-induced apoptosis, but had no such effect on S or G2M cells. However, no significant rise in the intracellular level of ROS upon treatment with etoposide was detected. The effects of etoposide were compared with the previously investigated effects of another topo2 inhibitor, mitoxantrone. The latter was seen to induce a maximal level of ATM-S1981P and γH2AX (partially abrogated by NAC) in G1-phase cells, but unlike etoposide, triggered apoptosis exclusively of S-phase cells. The data suggest that in addition to the generally accepted mechanism involving collisions of replication forks with the “cleavable complexes”, other mechanisms which appear to be different for etoposide vs. mitoxantrone, may contribute to formation of DSBs and to triggering of apoptosis.     

Synergistic interaction of proteasome and topoisomerase II inhibition in multiple myeloma

Multiple myeloma is a malignancy of terminally differentiated plasma cells and is incurable in the majority of the patients. Thus, novel effective treatment regimens are urgently needed. In this study, we examined the effects of co-treatment with proteasome-inhibitor bortezomib and topoisomerase II inhibitor etoposide in multiple myeloma cells lines OPM-2, RPMI-S and NCI-H929. Using the median effect method of Chou and Talalay, we evaluated the combination indices (CI) for simultaneous and sequential treatment schedules. In the sequential treatment schedule, we found strong synergistic effects in all three cell lines, even at low single-agent cytotoxicity levels. When cells were treated simultaneously with both drugs, the synergy was present but less pronounced than in the sequential treatment schedule. The synergistic effects observed in the co-treatment schedules were accompanied by an inhibition of anti-apoptotic effects that were induced by etoposide alone. Namely, bortezomib abrogated both etoposide-induced NF-κB activation and etoposide-induced bcl-2 up-regulation. Our data suggest that combining etoposide with bortezomib might be useful for cancer treatment, as bortezomib potentially inhibits counter-regulatory mechanisms of tumor cells, which are induced by topoisomerase II inhibition and which may contribute to acquired chemoresistance.     

Prostate cancer stem-like cells proliferate slowly and resist etoposide-induced cytotoxicity via enhancing DNA damage response

Despite the development of chemoresistance as a major concern in prostate cancer therapy, the underlying mechanisms remain elusive. In this report, we demonstrate that DU145-derived prostate cancer stem cells (PCSCs) progress slowly with more cells accumulating in the G1 phase in comparison to DU145 non-PCSCs. Consistent with the important role of the AKT pathway in promoting G1 progression, DU145 PCSCs were less sensitive to growth factor-induced activation of AKT in comparison to non-PCSCs. In response to etoposide (one of the most commonly used chemotherapeutic drugs), DU145 PCSCs survived significantly better than non-PCSCs. In addition to etoposide, PCSCs demonstrated increased resistance to docetaxel, a taxane drug that is commonly used to treat castration-resistant prostate cancer. Etoposide produced elevated levels of γH2AX and triggered a robust G2/M arrest along with a coordinated reduction of the G1 population in PCSCs compared to non-PCSCs, suggesting that elevated γH2AX plays a role in the resistance of PCSCs to etoposide-induced cytotoxicity. We have generated xenograft tumors from DU145 PCSCs and non-PCSCs. Consistent with the knowledge that PCSCs produce xenograft tumors with more advanced features, we were able to demonstrate that PCSC-derived xenograft tumors displayed higher levels of γH2AX and p-CHK1 compared to non-PCSC-produced xenograft tumors. Collectively, our research suggests that the elevation of DNA damage response contributes to PCSC-associated resistance to genotoxic reagents.     

Sulforaphane controls TPA-induced MMP-9 expression through the NF-κB signaling pathway,but not AP-1, in MCF-7 breast cancer cells

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells. [BMB Reports 2013; 46(4): 201-206]     

UV-induced apoptosis is mediated independent of caspase-9 in MCF-7 cells: a model for cytochrome c resistance

The importance of the mitochondria in UV-induced apoptosis has become increasingly apparent. Following DNA damage cytochrome c and other pro-apoptotic factors are released from the mitochondria, allowing for formation of the apoptosome and subsequent cleavage and activation of caspase-9. Active caspase-9 then activates downstream caspases-3 and/or -7, which in turn cleave poly(ADP)-ribose polymerase (PARP) and other down-stream targets, resulting in apoptosis. In an effort to understand the mechanisms of Akt-mediated cell survival in breast cancer, we studied the effects of insulin-like growth factor (IGF)-I treatment on UV-treated MCF-7 human breast cancer cells. Apoptosis was induced in MCF-7 cells after UV treatment, as measured by caspase-7 and PARP cleavage, and IGF-I co-treatment protected against this response. Surprisingly caspase-9 cleavage was unchanged with UV and/or IGF-I treatment. Using MCF-7 cells overexpressing caspase-3 we have shown that resistance of caspase-9 to cleavage was not altered by the expression of caspase-3. Furthermore, overexpression of caspase-9 did not enhance PARP or caspase-7 cleavage after UV treatment. Because caspase-8 was activated with UV treatment alone, we believe that UV-induced apoptosis in MCF-7 cells occurs independently of cytochrome c and caspase-9, supporting the existence of a cytoplasmic inhibitor of cytochrome c in MCF-7 cells. We anticipate that such inhibitors may be overexpressed in cancer cells, allowing for treatment resistance.     

Downregulation of Choline Kinase-Alpha Enhances Autophagy in Tamoxifen-Resistant Breast Cancer Cells

Choline kinase-α (Chk-α) and autophagy have gained much attention, as they relate to the drug-resistance of breast cancer. Here, we explored the potential connection between Chk-α and autophagy in the mechanisms driving to tamoxifen (TAM) resistance, in estrogen receptor positive (ER+) breast cancer cells (BCCs). Human BCC lines (MCF-7 and TAM-resistant MCF-7 (MCF-7/TAM) cells) were used. Chk-α expression and activity was suppressed by the transduction of shRNA (shChk-α) with lentivirus and treatment with CK37, a Chk-α inhibitor. MCF-7/TAM cells had higher Chk-α expression and phosphocholine levels than MCF-7 cells. A specific downregulation of Chk-α by the transduction of shChk-α exhibited a significant decrease in phosphocholine levels in MCF-7 and MCF-7/TAM cells. The autophagy-related protein, cleaved microtubule-associated protein light chain 3 (LC3) and autophagosome-like structures were significantly increased in shChk-α-transduced or CK37-treated MCF-7 and MCF-7/TAM cells. The downregulation of Chk-α attenuated the phosphorylation of AKT, ERK1/2, and mTOR in both MCF-7 and MCF-7/TAM cells. In MCF-7 cells, the downregulation of Chk-α resulted in an induction of autophagy, a decreased proliferation ability and an activation of caspase-3. In MCF-7/TAM cells, despite a significant decrease in proliferation ability and an increase in the percentage of cells in the G0/G1 phase of the cell cycle, the downregulation of Chk-α did not induced caspase-dependent cell death and further enhanced autophagy and G0/G1 phase arrest. An autophagy inhibitor, methyladenine (3-MA) induced death and attenuated the level of elevated LC3 in MCF-7/TAM cells. Elucidating the interplay between choline metabolism and autophagy will provide unique opportunities to identify new therapeutic targets and develop novel treatment strategies that preferentially target TAM-resistance.     

Interaction of vitamin D analogs with signaling pathways leading to active cell death in breast cancer cells

Induction of apoptosis is a feature of the anti-tumor effects of certain vitamin D analogs. The aim of this study was to identify if common effectors are involved in cell death mediated by serum starvation, vitamin D analogs and tumor necrosis factor (TNF) alpha in 3 human breast cancer cell lines: MCF-7, T47-D and Hs578T. Incubation of cells in serum-free medium induced apoptosis as assessed by loss of cell viability and increased DNA fragmentation. Addition of IGF-I (30 ng/ml) protected against loss of cell viability in MCF-7 cells and co-treatment with two synthetic analogs (CB1093 and EB1089, 50 nM for 4 days) prevented these anti-apoptotic effects of IGF-I. Pretreatment of MCF-7 and Hs578T cells with the vitamin D analogs substantially potentiated the cytotoxic effects of TNFalpha. This cytokine was not cytotoxic for T47-D cells but co-incubation with CB1093 led to loss of cell viability. Potentiation by CB1093 of TNFalpha-induced apoptosis in MCF-7 cells was accompanied by increased activation of cytosolic phospholipase A2 and arachidonic acid release, which was partially inhibited by AACOCF3, a specific cPLA2 inhibitor. The broad-spectrum caspase inhibitor z-VAD-fmk prevented TNFalpha but not CB1093 mediated cell death and activation of cPLA2. Serum starvation induced apoptosis was accompanied by cPLA2 activation, which was inhibited by IGF-I and by z-VAD-fmk. However, the ability of these agents to suppress cPLA2 activation was abrogated by co-treatment with CB1093, suggesting a role for arachidonic acid release in the caspase-independent mechanism by which vitamin D analogs prevent the protective effects of IGF-I on breast cancer cell survival.     

Curcumin Improves the Tumoricidal Effect of Mitomycin C by Suppressing ABCG2 Expression in Stem Cell-Like Breast Cancer Cells

Cancer cells with stem cell–like properties contribute to the development of resistance to chemotherapy and eventually to tumor relapses. The current study investigated the potential of curcumin to reduce breast cancer stem cell (BCSC) population for sensitizing breast cancer cells to mitomycin C (MMC) both in vitro and in vivo. Curcumin improved the sensitivity of paclitaxel, cisplatin, and doxorubicin in breast cancer cell lines MCF-7 and MDA-MB-231, as shown by the more than 2-fold decrease in the half-maximal inhibitory concentration of these chemotherapeutic agents. In addition, curcumin sensitized the BCSCs of MCF-7 and MDA-MB-231 to MMC by 5- and 15-fold, respectively. The BCSCs could not grow to the fifth generation in the presence of curcumin and MMC. MMC or curcumin alone only marginally reduced the BCSC population in the mammospheres; however, together, they reduced the BCSC population in CD44⁺CD24^−/low cells by more than 75% (29.34% to 6.86%). Curcumin sensitized BCSCs through a reduction in the expression of ATP-binding cassette (ABC) transporters ABCG2 and ABCC1. We demonstrated that fumitremorgin C, a selective ABCG2 inhibitor, reduced BCSC survival to a similar degree as curcumin did. Curcumin sensitized breast cancer cells to chemotherapeutic drugs by reducing the BCSC population mainly through a reduction in the expression of ABCG2.     

Withaferin A-induced apoptosis in human breast cancer cells is mediated by reactive oxygen species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.