Antineoplastic Effects of α-Santalol on Estrogen Receptor-Positive and Estrogen Receptor-Negative Breast Cancer Cells through Cell Cycle Arrest at G2/M Phase and Induction of Apoptosis

Anticancer efficacy and the mechanism of action of α-santalol, a terpenoid isolated from sandalwood oil, were investigated in human breast cancer cells by using p53 wild-type MCF-7 cells as a model for estrogen receptor(ER)-positive and p53 mutated MDA-MB-231 cells as a model for ER-negative breast cancer. α-Santalol inhibited cell viability and proliferation in a concentration and time-dependent manner in both cells regardless of their ER and/or p53 status. However, α-santalol produced relatively less toxic effect on normal breast epithelial cell line, MCF-10A. It induced G2/M cell cycle arrest and apoptosis in both MCF-7 and MDA-MB-231 cells. Cell cycle arrest induced by α-santalol was associated with changes in the protein levels of BRCA1, Chk1, G2/M regulatory cyclins, Cyclin dependent kinases (CDKs), Cell division cycle 25B (Cdc25B), Cdc25C and Ser-216 phosphorylation of Cdc25C. An up-regulated expression of CDK inhibitor p21 along with suppressed expression of mutated p53 was observed in MDA-MB-231 cells treated with α-santalol. On the contrary, α-santalol did not increase the expression of wild-type p53 and p21 in MCF-7 cells. In addition, α-santalol induced extrinsic and intrinsic pathways of apoptosis in both cells with activation of caspase-8 and caspase-9. It led to the activation of the executioner caspase-6 and caspase-7 in α-santalol-treated MCF-7 cells and caspase-3 and caspase-6 in MDA-MB-231 cells along with strong cleavage of poly(ADP-ribose) polymerase (PARP) in both cells. Taken together, this study for the first time identified strong anti-neoplastic effects of α-santalol against both ER-positive and ER-negative breast cancer cells.     

Different anticancer effects of Saxifragifolin A on estrogen receptor-positive and estrogen receptor-negative breast cancer cells

BackgroundBreast cancer is the leading cause of cancer-related death among women worldwide. For treating breast cancer, numerous natural products have been considered as chemotherapeutic drugs.Hypothesis/purposeThe present study aims to investigate the apoptotic effect of Saxifragifolin A (Saxi A) isolated from Androsace umbellata in two different human breast cancer cells which are ER-positive MCF-7 cells and ER-negative MDA-MB-231 cells, and examine the molecular basis for its anticancer actions.Study designThe inhibitory effects of Saxi A on cell survival were examined in MCF-7 cells and MDA-MB-231 cells in vitro.MethodsMTT assays, Annexin V/PI staining analysis, ROS production assay, Hoechst33342 staining and Western blot analysis were performed.ResultsOur results showed that MDA-MB-231 cells were more sensitive to Saxi A-induced apoptosis than MCF-7 cells. Saxi A induced apoptosis in MDA-MB-231 cells through ROS-mediated and caspase-dependent pathways, whereas treatment with Saxi A induced apoptosis in MCF-7 cells in a caspase-independent manner. In spite of Saxi A-induced activation of MAPKs in both breast cancer cell lines, only p38 MAPK and JNK mediated Saxi A-induced apoptosis. In addition, cell survival of shERα-transfected MCF-7 cells was decreased, while MDA-MB-231 cells that overexpress ERα remained viable.ConclusionSaxi A inhibits cell survival in MCF-7 cells and MDA-MB-231 cells through different regulatory pathway, and ERα status appears to be important for regulating Saxi A-induced apoptosis in breast cancer cells. Thus, Saxi A may have a potential therapeutic use for treating breast cancer.     

Palladium(II) saccharinate complexes with bis(2-pyridylmethyl)amine induce cell death by apoptosis in human breast cancer cells in vitro

The outcomes of breast cancer patients are still poor although new compounds have recently been introduced into the clinic. Therefore, novel chemical approaches are required. In the present study, palladium(II) and corresponding platinum(II) complexes containing bis(2-pyridylmethyl)amine (bpma) and saccharine were synthesized and tested against human breast cancer cell lines, MCF-7 and MDA-MB-231, in vitro. Cytotoxicity was first screened by the MTT assay and the results were further confirmed by the ATP assay. The palladium complexes 1 and 3 yielded stronger cytotoxicity than the corresponding platinum complexes 2 and 4 at the same doses. The palladium complex 3 was found to be the most cytotoxic one. Therefore, a more comprehensive study was carried out with this complex only. The mode of cell death was determined morphologically under fluorescent microscope and biochemically with detection of active caspase-3 and PARP cleavage by Western blot. Changes in apoptosis-related gene expressions were measured with qPCR. It was demonstrated that complex 3 caused cell death by apoptosis determined by fluorescence imaging and Western blot. As a sign of apoptosis, PARP was cleaved in both of the cell lines. In addition, caspase-3 was cleaved in MDA-MB-231 cells while this cleavage was not observed in MCF-7. The results show that the complex 3 is a promising anti-cancer compound against breast cancer with an IC₅₀ value of 3.9 μM for MCF-7 and 4.2 μM for MDA-MB-231 cells, which warrants further animal experiments.     

Different effects of genistein on molecular markers related to apoptosis in two phenotypically dissimilar breast cancer cell lines

The association between consumption of genistein-containing soybean products and lower risk of breast cancer suggests a cancer chemopreventive role for genistein. Consistent with this suggestion, exposing cultured human breast cancer cells to genistein inhibits cell proliferation, although this is not completely understood. To better understand how genistein works, the ability of genistein to induce apoptosis was compared in phenotypically dissimilar MCF-7 and MDA-MB-231 human breast cancer cells that express the wild-type and mutant p53 gene, respectively. After 6 days of incubation with 50 microM genistein, MCF-7 but not MDA-MB-231 cells, showed morphological signs of apoptosis. Marginal proteolytic cleavage of poly-(ADP-ribose)-polymerase and significant DNA fragmentation were also detected in MCF-7 cells. In elucidating these findings, it was determined that after 2 days of incubation with genistein, MCF-7 but not MDA-MB-231 cells, had significantly higher levels of p53. Accordingly, the expression of certain proteins modulated by p53 was studied next. Levels of p21 increased in both of the genistein-treated cell lines, suggesting that p21 gene expression was activated but in a p53-independent manner, whereas no significant changes in levels of the pro-apoptotic protein, Bax, were found. In MCF-7 cells, levels of the anti-apoptotic protein, Bcl-2, decreased slightly at 18-24 h but then increased considerably after 48 h. Hence, the Bax:Bcl-2 ratio initially increased but later decreased. These data suggest that at the genistein concentration tested, MCF-7 cells in contrast to MDA-MB-231 cells were sensitive to the induction of apoptosis by genistein, but Bax and Bcl-2 did not play clear roles.     

Anti-Proliferative Effects of Evodiamine on Human Breast Cancer Cells

Endocrine sensitivity, assessed by the expression of estrogen receptor (ER), has long been the predict factor to guide therapeutic decisions. Tamoxifen has been the most successful hormonal treatment in endocrine-sensitive breast cancer. However, in estrogen-insensitive cancer tamoxifen showed less effectiveness than in estrogen-sensitive cancer. It is interesting to develop new drugs against both hormone-sensitive and insensitive tumor. In this present study we examined anticancer effects of evodiamine extracted from the Chinese herb, Evodiae fructus, in estrogen-dependent and –independent human breast cancer cells, MCF-7 and MDA-MB-231 cells, respectively. Evodiamine inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a concentration-dependent manner with concentration of 1×10⁻⁶ and 1×10⁻⁵ M. Evodiamine also induced apoptosis via up-regulation of caspase 7 activation, PARP cleavage (Bik and Bax expression). The expression of ER α and β in protein and mRNA levels was down-regulated by evodiamine according to data from immunoblotting and RT-PCR analysis. Overall, our results indicate that evodiamine mediates degradation of ER and induces caspase-dependent pathway leading to inhibit proliferation of breast cancer cell lines. It suggests that evodiamine may in part mediate through ER-inhibitory pathway to inhibit breast cancer cell proliferation.     

Artemisia absinthium (AA): a novel potential complementary and alternative medicine for breast cancer

Natural products have become increasingly important in pharmaceutical discoveries, and traditional herbalism has been a pioneering specialty in biomedical science. The search for effective plant-derived anticancer agents has continued to gain momentum in recent years. The present study aimed to investigate the role of crude extracts of the aerial parts of Artemisia absinthium (AA) extract in modulating intracellular signaling mechanisms, in particular its ability to inhibit cell proliferation and promote apoptosis in a human breast carcinoma estrogenic-unresponsive cell line, MDA-MB-231, and an estrogenic-responsive cell line, MCF-7. Cells were incubated with various concentrations of AA, and anti-proliferative activity was assessed by MTT assays, fluorescence microscopy after propidium iodide staining, western blotting and cell cycle analysis. Cell survival assays indicated that AA was cytotoxic to both MDA-MB-231 and MCF-7 cells. The morphological features typical of nucleic staining and the accumulation of sub-G1 peak revealed that the extract triggered apoptosis. Treatment with 25 μg/mL AA resulted in activation of caspase-7 and upregulation of Bad in MCF-7 cells, while exposure to 20 μg/mL AA induced upregulation of Bcl-2 protein in a time-dependent response in MDA-MB-231 cells. Both MEK1/2 and ERK1/2 was inactivated in both cell lines after AA treatment in a time-dependent manner. These results suggest that AA-induced anti-proliferative effects on human breast cancer cells could possibly trigger apoptosis in both cell lines through the modulation of Bcl-2 family proteins and the MEK/ERK pathway. This might lead to its possible development as a therapeutic agent for breast cancer following further investigations.     

Benzyl isothiocyanate causes FoxO1-mediated autophagic death in human breast cancer cells

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, inhibits growth of breast cancer cells but the mechanisms underlying growth inhibitory effect of BITC are not fully understood. Here, we demonstrate that BITC treatment causes FoxO1-mediated autophagic death in cultured human breast cancer cells. The BITC-treated breast cancer cells (MDA-MB-231, MCF-7, MDA-MB-468, BT-474, and BRI-JM04) and MDA-MB-231 xenografts from BITC-treated mice exhibited several features characteristic of autophagy, including appearance of double-membrane vacuoles (transmission electron microscopy) and acidic vesicular organelles (acridine orange staining), cleavage of microtubule-associated protein 1 light chain 3 (LC3), and/or suppression of p62 (p62/SQSTM1 or sequestosome 1) expression. On the other hand, a normal human mammary epithelial cell line (MCF-10A) was resistant to BITC-induced autophagy. BITC-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was partially but statistically significantly attenuated in the presence of autophagy inhibitors 3-methyl adenine and bafilomycin A1. Stable overexpression of Mn-superoxide dismutase, which was fully protective against apoptosis, conferred only partial protection against BITC-induced autophagy. BITC treatment decreased phosphorylation of mTOR and its downstream targets (P70s6k and 4E-BP1) in cultured MDA-MB-231 and MCF-7 cells and MDA-MB-231 xenografts, but activation of mTOR by transient overexpression of its positive regulator Rheb failed to confer protection against BITC-induced autophagy. Autophagy induction by BITC was associated with increased expression and acetylation of FoxO1. Furthermore, autophagy induction and cell growth inhibition resulting from BITC exposure were significantly attenuated by small interfering RNA knockdown of FoxO1. In conclusion, the present study provides novel insights into the molecular circuitry of BITC-induced cell death involving FoxO1-mediated autophagy.     

Novel quinuclidinone derivatives induced apoptosis in human breast cancer via targeting p53

Small molecules that can target human cancers have been highly sought to increase the anticancer efficacy, the present work describes the design and synthesis of novel series of five quinuclidinone derivatives (2a-2e). Their anticancer activities were investigated against breast cancer cells MCF-7, MDA-MB-231 breast cancer cells harboring mutant p53 and normal breast counterpart MCF-12a. Derivative 2e reduced proliferation of MCF-7 and MCF-12a while it has no effect on MDA-MB-231. Derivative 2e induced apoptosis in MCF-7 cells which is further confirmed by TUNEL assay and it reduced the percentage of cell in G2/M phase as confirmed by increased expression of cyclin B and reduced expression of cyclin D1. Derivative 2e reduced expression levels of Mdm2, Akt and ERK1/2 by and increased expression level of p53. Moreover, the apoptosis induction by 2e was also inhibited by PFT-α as evidenced by non-significant induction of apoptosis after treatment of MCF-7 cells with both derivative 2e and PFT-α. In addition, docking study reveals that derivative 2e has a binding pattern close to the pattern observed in the structure of the lead fragment 5,6-dimethoxy-2-methylbenzothiazole bound to T-p53C-Y220C. The above findings demonstrate that derivative 2e induces apoptosis in MCF-7 cells via targeting p53 which merits further development.     

Curcumin-induced Aurora-A suppression not only causes mitotic defect and cell cycle arrest but also alters chemosensitivity to anticancer drugs

Overexpression of oncoprotein Aurora-A increases drug resistance and promotes lung metastasis of breast cancer cells. Curcumin is an active anticancer compound in turmeric and curry. Here we observed that Aurora-A protein and kinase activity were reduced in curcumin-treated human breast chemoresistant nonmetastatic MCF-7 and highly metastatic cancer MDA-MB-231 cells. Curcumin acts in a similar manner to Aurora-A small interfering RNA (siRNA), resulting in monopolar spindle formation, S and G2/M arrest, and cell division reduction. Ectopic Aurora-A extinguished the curcumin effects. The anticancer effects of curcumin were enhanced by Aurora-A siRNA and produced additivity and synergism effects in cell division and monopolar phenotype, respectively. Combination treatment with curcumin overrode the chemoresistance to four Food and Drug Administration (FDA)-approved anticancer drugs (ixabepilone, cisplatin, vinorelbine, or everolimus) in MDA-MB-231 cells, which was characterized by a decrease in cell viability and the occurrence of an additivity or synergy effect. Ectopic expression of Aurora-A attenuated curcumin-enhanced chemosensitivity to these four tested drugs. A similar benefit of curcumin was observed in MCF-7 cells treated with ixabepilone, the primary systemic therapy to patients with invasive breast cancer (stages IIA–IIIB) before surgery. Antagonism effect was observed when MCF-7 cells were treated with curcumin plus cisplatin, vinorelbine or everolimus. Curcumin-induced enhancement in chemosensitivity was paralleled by significant increases (additivity or synergy effect) in apoptosis and cell cycle arrest at S and G2/M phases, the consequences of Aurora-A inhibition. These results suggest that a combination of curcumin with FDA-approved anticancer drugs warrants further assessment with a view to developing a novel clinical treatment for breast cancer.     

Critical role of p53 upregulated modulator of apoptosis in benzyl isothiocyanate-induced apoptotic cell death

Benzyl isothiocyanate (BITC), a constituent of edible cruciferous vegetables, decreases viability of cancer cells by causing apoptosis but the mechanism of cell death is not fully understood. The present study was undertaken to determine the role of Bcl-2 family proteins in BITC-induced apoptosis using MDA-MB-231 (breast), MCF-7 (breast), and HCT-116 (colon) human cancer cells. The B-cell lymphoma 2 interacting mediator of cell death (Bim) protein was dispensable for proapoptotic response to BITC in MCF-7 and MDA-MB-231 cells as judged by RNA interference studies. Instead, the BITC-treated MCF-7 and MDA-MB-231 cells exhibited upregulation of p53 upregulated modulator of apoptosis (PUMA) protein. The BITC-mediated induction of PUMA was relatively more pronounced in MCF-7 cells due to the presence of wild-type p53 compared with MDA-MB-231 with mutant p53. The BITC-induced apoptosis was partially but significantly attenuated by RNA interference of PUMA in MCF-7 cells. The PUMA knockout variant of HCT-116 cells exhibited significant resistance towards BITC-induced apoptosis compared with wild-type HCT-116 cells. Attenuation of BITC-induced apoptosis in PUMA knockout HCT-116 cells was accompanied by enhanced G2/M phase cell cycle arrest due to induction of p21 and down regulation of cyclin-dependent kinase 1 protein. The BITC treatment caused a decrease in protein levels of Bcl-xL (MCF-7 and MDA-MB-231 cells) and Bcl-2 (MCF-7 cells). Ectopic expression of Bcl-xL in MCF-7 and MDA-MB-231 cells and that of Bcl-2 in MCF-7 cells conferred protection against proapoptotic response to BITC. Interestingly, the BITC-treated MDA-MB-231 cells exhibited induction of Bcl-2 protein expression, and RNA interference of Bcl-2 in this cell line resulted in augmentation of BITC-induced apoptosis. The BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with the induction of PUMA protein in the tumor. In conclusion, the results of the present study indicate that Bim-independent apoptosis by BITC in cancer cells is mediated by PUMA.     

Synthesis and biological evaluation of a cyclic ether fluorinated noscapine analog

We present here a novel semi-synthetic cyclic ether fluorinated noscapine analog (CEFNA) that shows potent antiproliferative and anticancer activity in both hormone-responsive (MCF-7) and hormone non-responsive (MDA-MB-231) breast cancer cells. Interestingly, it is also effective against MCF-7/Adr, an adriamycin-resistant variant of MCF-7 cells. Immunofluorescence experiments showed numerous micronuclei, indicative of apoptotic cell death triggered by this novel analog. Mechanistically, CEFNA exerts a strong antimitotic effect as revealed by cell-cycle studies that show a dose-dependent increase in G2/M population preceding a rising sub-G1 population, suggesting apoptosis.     

Role of mitochondria in tamoxifen-induced rapid death of MCF-7 breast cancer cells

Tamoxifen (Tam) is widely used in chemotherapy of estrogen receptor-positive breast cancer. It inhibits proliferation and induces apoptosis of breast cancer cells by estrogen receptor-dependent modulation of gene expression, but recent reports have shown that Tam (especially at pharmacological concentrations) has also rapid nongenomic effects. Here we studied the mechanisms by which Tam exerts rapid effects on breast cancer cell viability. In serum-free medium 5–7 μM Tam induced death of MCF-7 and MDA-MB-231 cells in a time-dependent manner in less than 60 min. This was associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane potential and an increase in production of reactive oxygen species (ROS). This suggests that disruption of mitochondrial function has a primary role in the acute death response of the cells. Accordingly, bongkrekic acid, an inhibitor of mitochondrial permeability transition, was able to protect MCF-7 cells against Tam. Rapid cell death induction by Tam was not associated with immediate activation of caspase-9 or cleavage of poly (ADP-ribose) polymerase. It was not blocked by the caspase inhibitor z-Val-Ala-Asp-fluoromethylketone either. Diphenylene ionodium (DPI), an inhibitor of NADPH oxidase, was able to prevent Tam-induced cell death but not cytochrome c release, which suggests that ROS act distal to cytochrome c. The pure antiestrogen ICI 182780 (1 μM) could partly oppose the effect of Tam in estrogen receptor positive MCF-7 cells, but not in estrogen receptor negative MDA-MB-231 cells. Pre-culturing MCF-7 cells in the absence of 17β-estradiol (E₂) or in the presence of a low Tam concentration (1 μM) made the cells even more susceptible to rapid death induction by 5 or 7 μM Tam. This effect was associated with decreased levels of the anti-apoptotic proteins Bcl-X_L and Bcl-2. In conclusion, our results demonstrate induction of a rapid mitochondrial cell death program in breast cancer cells at pharmacological concentrations of Tam, which are achievable in tumor tissue of Tam-treated breast cancer patients. These mechanisms may contribute to the ability of Tam therapy to induce death of breast cancer cells.     

Synergistic enhancement of apoptosis by coralyne and paclitaxel in combination on MDA-MB-231 a triple-negative breast cancer cell line

Triple-negative breast cancer (TNBC) is the most outrageous subtype of breast cancer. Emphasizing the urge of new approach in cancer therapy, combinational drug therapy may be proven as an effective strategy. In our previous study, we reported that coralyne (COR) with paclitaxel (PTX) efficiently decreases the proliferation of MDA-MB-231 compared with MCF-7 cell line. Thus, we studied the effect of COR and PTX in combination on apoptosis of MDA-MB-231 cell line. In silico results demonstrated that COR intercalates DNA at a minor groove. In vitro approaches revealed that in combination (COR and PTX) increases the efficacy of apoptosis in MDA-MB-231 cell line by a significant increase in G1/S phase arrest, DNA fragmentation, and change in mitochondria membrane potential. The expression of ATM and ATR a serine/threonine-protein kinase, ataxia telangiectasia and Rad3-related protein were depleted with an increase in time from 24 to 48 hours in concurrent with increased levels of γH2AX indicating that DNA damage routes cells to enter apoptosis. This was confirmed by high levels of caspase-3 and cytochrome c. Also, the decrease in the expression levels of matrix metalloproteinase-9 confirmed the antimetastatic efficacy of COR + PTX. The present study indicates that the synergistic effect of COR and PTX can enhance apoptosis in MDA-MB-231 cell line and may be proven as a potential anticancer therapy against TNBC.     

Amphotericin B potentiates the anticancer activity of doxorubicin on the MCF-7 breast cancer cells

Despite the improvements in cancer treatment, breast cancer still remains the second most common cause of death from cancer in women. Doxorubicin (DOXO) is widely used for cancer treatment. However, drug resistance limits the treatment outcome. Here, we investigated the toxicity of DOXO in combination with an antifungal agent amphotericin B (AmB) against the MCF-7 breast cancer cell line. The cell viability was measured using MTT assay. The apoptosis was studied by caspase-8 and caspase-9 activity measurements and DNA fragmentation was investigated by TUNEL assay. The combination of two drugs significantly increased the apoptotic index and the caspase-8 and caspase-9 activities in comparison to DOXO-treated cells. Our finding showed that pre-treatment of MCF-7 cells with AmB synergistically exerted the anticancer effect of DOXO through the caspase-dependent apoptosis manner.     

Radiosensitizing effect of conjugated linoleic acid on tumor cells MCF-7 and MDA-MB-231

Apoptotic pathways in breast cancer cells are frequently altered, reducing the efficiency of radiotherapy. Conjugated linoleic acid (CLA), known to trigger apoptosis, was tested as radiosensitizer in breast cancer cells MCF-7 and MDA-MB-231. The CLA-mix, made up of the isomers CLA-9cis 11trans and CLA-10trans 12cis, was compared to three purified isomers, i.e., the CLA-9cis 11cis, CLA-9cis 11trans, and CLA-10trans 12cis. Using the apoptotic marker YO-PRO-1, the CLA-9cis 11cis at 50 micro mol/L turned out to be the best apoptotic inducer leading to a 10-fold increase in MCF-7 cells and a 2,5-fold increase in MDA-MB-231 cells, comparatively to the CLA-mix. Contrary to previous studies on colorectal and prostate cancer cells, CLA-10trans 12cis does not lead to an apoptotic response on breast cancer cell lines MCF-7 and MDA-MB-231. Our results also suggest that the main components of the CLA-mix (CLA-9cis 11trans and CLA-10trans 12cis) are not involved in the induction of apoptosis in the breast cancer cells studied. A dose of 5 Gy did not induce apoptosis in MCF-7 and MDA-MB-231 cells. The addition of CLA-9cis 11cis or CLA-mix has allowed us to observe a radiation-induced apoptosis, with the CLA-9cis 11cis being about 8-fold better than the CLA-mix. CLA-9cis 11cis turned out to be the best radiosensitizer, although the isomers CLA-9cis 11trans and CLA-10trans 12cis have also reduced the cell survival following irradiation, but using a mechanism not related to apoptosis. In conclusion, the radiosensitizing property of CLA-9cis 11cis supports its potential as an agent to improve radiotherapy against breast carcinoma.     

Metformin reverses multidrug resistance and epithelial–mesenchymal transition (EMT) via activating AMP-activated protein kinase (AMPK) in human breast cancer cells

Breast cancer is the most frequently diagnosed tumor type and the primary leading cause of cancer deaths in women worldwide and multidrug resistance is the major obstacle for breast cancer treatment improvement. Emerging evidence suggests that metformin, the most widely used antidiabetic drug, resensitizes and cooperates with some anticancer drugs to exert anticancer effect. However, there are no data regarding the reversal effect of metformin on chemoresistance in breast cancer. In the present study, we investigated the resistance reversal effect of metformin on acquired multidrug-resistant breast cancer cells MCF-7/5-Fu derived from MCF-7 breast cancer cells and innate multidrug-resistant MDA-MB-231 breast cancer cells, and we found that metformin resensitized MCF7/5-FU and MDA-MB-231 to 5-fluorouracil (5-FU), adriamycin, and paclitaxel. We also observed that metformin reversed epithelial–mesenchymal transition (EMT) phenotype and decreased the invasive capacity of MCF7/5-FU and MDA-MB-231 cells. However, there were no significant changes upon metformin-treated MCF7 cells. Moreover, we found metformin treatment activated AMPK signal pathway in MCF7/5-FU and MDA-MB-231 cells and compound C, the AMPK inhibitor, could partly abolish the resensitization and EMT reversal effect of metformin. To the best of our knowledge, we are the first to report that metformin can resensitize multidrug-resistant breast cancer cells due to activating AMPK signal pathway. Our study will help elucidate the mechanism of chemoresistance and establish new strategies of chemotherapy for human breast cancer.     

The Oncogene Metadherin Modulates the Apoptotic Pathway Based on the Tumor Necrosis Factor Superfamily Member TRAIL (Tumor Necrosis Factor-related Apoptosis-inducing Ligand) in Breast Cancer

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.     

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.     

Development of methotrexate proline prodrug to overcome resistance by MDA-MB-231 cells

The resistance to methotrexate by a number of cancer cells such as breast cancer cell-line MDA-MB-231 due to poor permeability renders it less effective as an anticancer agent for these cells. Proline prodrug of methotrexate (Pro-MTX) was designed as a substrate of prolidase which is specific for imido bond of dipeptide containing proline and expected to penetrate MDA-MB-231 cells more efficiently. The prodrug was synthesized by solid-phase peptide synthesis method and examined as a substrate of pure prolidase as well as cell homogenate. The cytotoxicity against MDA-MB-231 and non-methotrexate resistant breast cancer cell line, MCF-7 was also examined by XTT assay. The results showed that Pro-MTX was a substrate of prolidase. It was also shown that the prodrug could be converted to parent drug methotrexate in Caco-2 and HeLa cell homogenate. When tested with Caco-2 and MCF-7 cells, Pro-MTX showed weaker cytotoxicity compared with methotrexate. But for methotrexate resistant MDA-MB-231 cells, Pro-MTX showed stronger activity than methotrexate. The results indicated that the proline prodrug of methotrexate may overcome the resistance of human breast cancer cells in culture.     

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.