Overcoming radioresistance of breast cancer cells with MAP4K4 inhibitors.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) has recently emerged as a promising therapeutic target in cancer. In this study, we explored the biological function of MAP4K4 in radioresistant breast cancer cells using two MAP4K4 inhibitors, namely PF06260933 and GNE-495. Radioresistant SR and MR cells were established by exposing SK-BR-3 and MCF-7 breast cancer cells to 48-70 Gy of radiation delivered at 4-5 Gy twice a week over 10 months. Surprisingly, although radioresistant cells were derived from two different subtypes of breast cancer cell lines, MAP4K4 was significantly elevated regardless of subtype. Inhibition of MAP4K4 with PF06260933 or GNE-495 selectively targeted radioresistant cells and improved the response to irradiation. Furthermore, MAP4K4 inhibitors induced apoptosis through the accumulation of DNA damage by inhibiting DNA repair systems in radioresistant cells. Notably, Inhibition of MAP4K4 suppressed the expressions of ACSL4, suggesting that MAP4K4 functioned as an upstream effector of ACSL4. This study is the first to report that MAP4K4 plays a crucial role in mediating the radioresistance of breast cancer by acting upstream of ACSL4 to enhance DNA damage response and inhibit apoptosis. We hope that our findings provide a basis for the development of new drugs targeting MAP4K4 to overcome radioresistance.

Oxidative tryptamine dimers from Corynebacterium durum directly target survivin to induce AIF-mediated apoptosis in cancer cells.

Accumulating evidence indicates that microbial communities in the human body crucially affect health through the production of chemical messengers. However, the relationship between human microbiota and cancer has been underexplored. As a result of a biochemical investigation of the commensal oral microbe, Corynebacterium durum, we identified the non-enzymatic transformation of tryptamine into an anticancer compound, durumamide A (1). The structure of 1 was determined using LC-MS and NMR data analysis as bis(indolyl)glyoxylamide, which was confirmed using one-pot synthesis and X-ray crystallographic analysis, suggesting that 1 is an oxidative dimer of tryptamine. Compound 1 displayed cytotoxic activity against various cancer cell lines with IC50 values ranging from 25 to 35 µM. A drug affinity responsive target stability assay revealed that survivin is the direct target protein responsible for the anticancer effect of 1, which subsequently induces apoptosis-inducing factor (AIF)-mediated apoptosis. Inspired by the chemical structure and bioactivity of 1, a new derivative, durumamide B (2), was synthesized using another indole-based neurotransmitter, serotonin. The anticancer properties of 2 were similar to those of 1; however, it was less active. These findings reinforce the notion of human microbiota-host interplay by showing that 1 is naturally produced from the human microbial metabolite, tryptamine, which protects the host against cancer.

Evaluation of Radiotherapy-Induced Systemic Antitumor Effects in Mice Bearing 4T1 Mouse Breast Cancer Cells.

Background: Recently, several clinical studies have reported that combination treatments of radiation therapy (RT) and immunotherapy in patients with multiple lesions can improve tumor regression at a distance from the irradiated site, known as the abscopal effect. However, when RT and immunotherapy are concurrently applied, it is hard to distinguish the pure systemic effects of RT from those of the immunotherapy drug. In this preclinical study, the authors investigated the systemic antitumor effects of RT alone according to fraction dose size and splitting schedules. Materials and Methods: 4T1 mouse breast cancer cells were implanted into the right and left sides of mammary gland fat pads of BALB/c mice, followed by irradiation with 6 Gy × 3, 8 Gy × 2, and 13 Gy × 1 fractions when the right-side tumors were palpable. Results: The different irradiation schedules produced similar antitumor effects in irradiated right-side tumors and unirradiated left-side tumors. However, 8 Gy × 2 and 13 Gy × 1 fractions exhibited better antimetastatic potential than that from irradiation using 6 Gy × 3 fractions. Furthermore, 8 Gy × 2 and 13 Gy × 1 fractions produced higher expressions of HMGB1 and lower expressions of the proinflammatory cytokines, IFN-γ, TNF-α, IL-6, and IL-1ß, from the irradiated tumor tissues. Conclusions: These findings suggest that 8 Gy × 2 and 13 Gy × 1 fractions can provide better systemic antitumor effects than 6 Gy × 3 fractions. The authors hope these results provide clues to optimize RT dose regimens to make the abscopal effect clinically more relevant in future combination treatments.

Acyl-CoA synthetase-4 mediates radioresistance of breast cancer cells by regulating FOXM1.

The development of radioresistance during radiotherapy is a major cause of tumor recurrence and metastasis. To provide new insights of the mechanisms underlying radioresistance, we established radioresistant cell lines derived from two different subtypes of breast cancer cells, HER2-positive SK-BR-3 and ER-positive MCF-7 breast cancer cells, by exposing cells to 48 ~ 70 Gy of radiation delivered at 4-5 Gy twice weekly over 9 ~ 10 months. The established radioresistant SK-BR-3 (SR) and MCF-7 (MR) cells were resistant not only to a single dose of radiation (2 Gy or 4 Gy) but also to fractionated radiation delivered at 2 Gy/day for 5 days. Furthermore, these cells exhibited tumor-initiating potential in vivo and high CD24-/CD44 + ratio. To identify novel therapeutic molecular targets, we analyzed differentially expressed genes in both radioresistant cell lines and found that the expression of ACSL4 was significantly elevated in both cell lines. Targeting ACSL4 improved response to irradiation and inhibited migration activities. Furthermore, inhibition of ACLS4 using ASCL4 siRNA or triacsin C suppressed FOXM1 expression, whereas inhibition of FOXM1 using thiostrepton did not affect ACSL4 expression. Targeting the ACSL4-FOXM1 signaling axis by inhibiting ASCL4 or FOXM1 overcame the radioresistance by suppressing DNA damage responses and inducing apoptosis. This is the first study to report that ACSL4 plays a crucial role in mediating the radioresistance of breast cancer by regulating FOXM1. We propose the ACSL4-FOXM1 signaling axis be considered a novel therapeutic target in radioresistant breast cancer and suggest treatment strategies targeting this signaling axis might overcome breast cancer radioresistance.

Cell cycle arrest-mediated cell death by morin in MDA-MB-231 triple-negative breast cancer cells.

BACKGROUND: Morin, a flavonoid extracted from Moraceace family and exhibits several pharmacological activities including anti-cancer activity. Although the anticancer activity of morin in breast cancer was estimated in some investigations, the pharmaceutical mechanism has not been fully elucidated. Therefore, we investigated to unveil the detail signaling pathway in morin-treated in MDA-MB-231 triple-negative breast cancer cells. METHODS: The cytotoxicity of morin in MDA-MB-231 cells was confirmed by sulforhodamine B (SRB) assay and colony formation assay. Flow cytometry was performed to examine the cell cycle and cell death patterns and the protein expression and phosphorylation were detected by western blotting. RESULTS: Our results showed that morin inhibited MDA-MB-231 cells proliferation in time and concentration-dependent manner. Morphological changes were observed when treated with various concentration of morin in MDA-MB-231 cells. In regard to protein expression, morin induced the phosphorylation of ERK and p-H2A.X and decreased the level of DNA repair markers, RAD51 and survivin. In addition, flow cytometry showed S and G2/M arrest by morin that was associated with the decrease in the protein expression of cyclin A2 and cyclin B1 and upregulation of p21. Interestingly, annexin V/PI staining result clearly showed that morin induced cell death without apoptosis. Furthermore, attenuated FoxM1 by morin was co-related with cell cycle regulators including p21, cyclin A2 and cyclin B1. CONCLUSION: Taken together, our study indicates that morin-induced cell death of MDA-MB-231 is caused by sustained cell cycle arrest via the induction of p21 expression by activation of ERK and repression of FOXM1 signaling pathways.

Tamoxifen overcomes the trastuzumab-resistance of SK-BR-3 tumorspheres by targeting crosstalk between cytoplasmic estrogen receptor α and the EGFR/HER2 signaling pathway.

Since trastuzumab-resistance remains a major obstacle to the successful treatment of HER2-positive breast cancer, a detailed understanding of the mechanisms responsible is required to direct future pharmacotherapeutic strategies. Recently, several studies have indicated that the quiescent natures of cancer stem cells contribute to treatment resistance and tumor recurrence. Thus, in this study, we investigated the mechanism underlying trastuzumab resistance in a quiescent cell population using tumorsphere cultures and explored better therapeutic strategies to overcome trastuzumab resistance in HER2-positive breast cancer patients. We observed that most cells in SK-BR-3 tumorspheres were quiescent, showing the accumulation of cells at the G0/G1 phase as compared to cells in monolayer culture. Furthermore, SK-BR-3 tumorspheres exhibited enhanced EGFR/HER2 signaling, which was incompletely inhibited by trastuzumab, and subsequently led to trastuzumab-resistance. Interestingly, cytoplasmic estrogen receptor α (ERα) expression was markedly elevated in tumorspheres and was associated with enhanced EGFR/HER2 signaling. Accordingly, inhibition of ERα with tamoxifen selectively targeted tumorspheres rather than cells in monolayer culture and overcame trastuzumab resistance in tumorspheres. Taken together, our findings indicate that crosstalk between cytoplasmic ERα and the HER2/EGFR signaling pathway can be considered a novel therapeutic target for quiescent cell populations within HER2-positive breast cancer and that simultaneous inhibition of ER and the EGFR/HER2 pathway may prevent trastuzumab resistance. We hope that these results provide a basis for the use of combinations of tamoxifen and trastuzumab in HER2-positive breast cancer patients.

Chaga mushroom extract induces autophagy via the AMPK-mTOR signaling pathway in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chaga mushrooms (Inonotus obliquus) are commonly used in traditional treatments in Eastern Europe and Asia due to their diverse pharmacological effects, including anti-tumor and immunologic effects. Thus, many cancer patients take Chaga mushrooms as a complementary medicine, even during chemotherapy or radiotherapy. However, few studies have investigated the effects or molecular targets of Chaga mushrooms in breast cancer. AIM OF THE STUDY: Herein, we examined the anticancer effects of Chaga mushrooms in different types of breast cancer cell lines, and explored the underlying molecular mechanism to better understand their effects and benefits. MATERIALS AND METHODS: Chaga mushroom extract (CME) was prepared by extracting Chaga mushrooms with 70% ethanol. The cytotoxic effects of CME were assessed by MTT assay and protein expressions were evaluated by western blotting. To evaluate in vivo anti-tumor effects of CME, CME (2 g/kg) was orally administered to 4T1 tumor-bearing BALB/c mice every other day over 30 days (15 administrations), and tumor sizes were measured. Silica gel column chromatography was used to fractionate CME, and major constituents responsible for cytotoxic effects of CME were identified by 1H/13C-NMR and LC-MS. RESULTS: CME inhibited the proliferation of 4T1 mouse breast cancer cells in a dose and time-dependent manner. The expression of LC3 and phosphorylation of AMPK were increased by CME, while the phosphorylation of mTOR, S6, and S6K1 were suppressed, suggesting that CME induced autophagy by activating AMPK and inhibiting mTOR signaling pathways. Consistent with its observed cytotoxic effect in vitro, CME effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition, inotodiol and trametenolic acid were identified as the major constituents responsible for the cytotoxic effects of CME on breast cancer cells. Moreover, inotodiol and trametenolic acid-enriched fractions both exhibited cytotoxic effects regardless of breast cancer cell subtypes and did not interfere with the cytotoxic effects of conventional drugs. CONCLUSIONS: Taken together, Chaga mushroom extract induced autophagy by activating AMPK and inhibiting the mTOR signaling pathway. Our data suggest Chaga mushrooms may be a beneficial complementary medicine for breast cancer patients.

A new function for MAP4K4 inhibitors during platelet aggregation and platelet-mediated clot retraction.

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is implicated in type 2 diabetes mellitus, insulin tolerance, inflammation, cancer, and atherosclerosis. We found that GNE 495 and PF 06260933 (both potent and selective MAP4K4 inhibitors) regulated human platelet activation. Immunoblotting revealed human platelets express MAP4K4, and that GNE 495 and PF 06260933 inhibited collagen-, ADP-, and thrombin-induced platelet aggregation and eventually suppressed granule release, TXA2 generation, integrin αIIbß3 activation, and clot retraction. In addition, both inhibitors elevated intracellular levels of cAMP, and coincubation with GNE 495 and aspirin or dipyridamole (a phosphodiesterase inhibitor) synergistically inhibited collagen-induced platelet aggregation and TXA2 generation. Moreover, both inhibitors phosphorylated VASP (ser157), IP3 receptor, and PKA and attenuated MAPK and PI3K/Akt/GSK3ß signaling pathways. This study is the first to demonstrate that MAP4K4 inhibitors reduce thrombus formation by inhibiting platelet activation. These findings also suggest MAP4K4 be considered an emerging target protein for the treatment of thrombosis.

Mistletoe Extract Targets the STAT3-FOXM1 Pathway to Induce Apoptosis and Inhibits Metastasis in Breast Cancer Cells.

Mistletoe extracts (Viscum album L.) have been widely used as complementary and alternative medicines for the treatment of cancer, and their cytotoxic effects have been reported on various types of cancer. However, the molecular targets of mistletoe extracts have not been well studied. Herein, we investigated molecules associated with the in vitro and in vivo anticancer effects of mistletoe extract using 4T1 murine breast cancer cells. Mistletoe extract induced apoptosis and inhibited the signal transducer and activator of transcription3 (STAT3) phosphorylation. This inhibition was accompanied by the downregulations of forkhead box M1 (FOXM1) and the DNA repair proteins, RAD51 and survivin. Mistletoe extract simultaneously increased the expression of the DNA damage marker proteins, phosphorylated H2A histone family member X (H2A.X), and phosphorylated p38. Furthermore, mistletoe extract effectively suppressed tumor growth in 4T1 tumor-bearing BALB/c mice. In addition to tumor growth inhibition, mistletoe extract inhibited lung metastasis in the tumor-bearing mice and cell invasiveness by downregulating the expressions of matrix metalloproteinases (MMPs), urokinase-type plasminogen activator (uPA), uPA receptor, and markers of epithelial-mesenchymal transition (snail and fibronectin). Taken together, our results suggest that mistletoe extract targets the STAT3-FOXM1 pathway for its cytotoxic effects, and that mistletoe extracts might be useful for the treatment of patients with cancers highly expressing the STAT3-FOXM1 pathway.

Arctigenin Enhances the Cytotoxic Effect of Doxorubicin in MDA-MB-231 Breast Cancer Cells.

Several reports have described the anti-cancer activity of arctigenin, a lignan extracted from Arctium lappa L. Here, we investigated the effect of arctigenin (ATG) on doxorubicin (DOX)-induced cell death using MDA-MB-231 human breast cancer cells. The results showed that DOX-induced cell death was enhanced by ATG/DOX co-treatment in a concentration-dependent manner and that this was associated with increased DOX uptake and the suppression of multidrug resistance-associated protein 1 (MRP1) gene expression in MDA-MB-231 cells. ATG enhanced DOX-induced DNA damage and decreased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and the expressions of RAD51 and survivin. Cell death caused by ATG/DOX co-treatment was mediated by the nuclear translocation of apoptosis inducing factor (AIF), reductions in cellular and mitochondrial Bcl-2 and Bcl-xL, and increases in mitochondrial BAX levels. However, caspase-3 and -7 did not participate in DOX/ATG-induced cell death. We also found that DOX/ATG-induced cell death was linked with activation of the p38 signaling pathway and suppressions of the phosphorylations and expressions of Akt and c-Jun N-terminal kinase. Taken together, these results show that ATG enhances the cytotoxic activity of DOX in MDA-MB-231 human breast cancer cells by inducing prolonged p21 expression and p38-mediated AIF-dependent cell death. In conclusion, our findings suggest that ATG might alleviate the side effects and improve the therapeutic efficacy of DOX.

Metformin selectively targets 4T1 tumorspheres and enhances the antitumor effects of doxorubicin by downregulating the AKT and STAT3 signaling pathways.

Recent studies have reported that metformin (Met), the first-line medication for the treatment of type 2 diabetes, exhibited anticancer and chemoprotective effects in diverse cancer cells. In this study, we investigated the effects of Met on the drug-resistance of 4T1 murine breast cancer tumorspheres (TS) and the mechanism responsible for its drug-resistance. 4T1 TS exhibited accumulations of cells at the G0/G1 phase compared with cells in monolayer culture, which suggested the majority of cells in TS were quiescent. Furthermore, it was identified that activations of the signal transducer and activator of transcription 3 (STAT3) and protein kinase B (AKT) signaling pathways in 4T1 TS conferred drug-resistance to doxorubicin (Dox) and lapatinib (Lapa). However, Met selectively targeted TS rather than cells in monolayer culture and increased the cytotoxic effect of Dox on TS by inhibiting activations of the STAT3 and AKT signaling pathways. These observations suggested that inhibitions of STAT3 and AKT underlie the selective cytotoxic effects of Met on TS. In addition, Met exhibited synergistic antitumor effects with Dox on 4T1 tumor-bearing BALB/c mice. Our findings suggest that combinations of Met and cytotoxic anticancer drugs may offer an advantage for treating drug-resistant breast cancer.

Deep sea water improves hypercholesterolemia and hepatic lipid accumulation through the regulation of hepatic lipid metabolic gene expression.

A high­fat diet or high­cholesterol diet (HCD) is a major cause of metabolic diseases, including obesity and diabetes; vascular diseases, including hypertension, stroke and arteriosclerosis; and liver diseases, including hepatic steatosis and cirrhosis. The present study aimed to evaluate the effects of deep sea water (DSW) on rats fed a HCD. DSW decreased HCD­induced increases in total cholesterol and low­density lipoprotein (LDL) cholesterol in the blood, and recovered high­density lipoprotein cholesterol. In addition, DSW decreased levels of liver injury markers, which were increased in response to HCD, including glutamate­oxaloacetate transaminase, glutamate­pyruvate transferase and alkaline phosphatase. Lower lipid droplet levels were observed in the livers of rats fed a HCD and treated with DSW at a hardness of 1,500, as compared with those in the HCD only group. Semi­quantitative reverse transcription­polymerase chain reaction (RT­PCR) revealed that mRNA expression levels of fatty acid synthase and sterol regulatory element binding protein­1c (SREBP­1c) in rats fed a HCD with DSW were lower compared with the HCD only group. Furthermore, quantitative RT­PCR revealed that DSW enhanced LDL receptor (LDLR) mRNA expression in a hardness­dependent manner. Combined, the results of the present study indicated that DSW may reduce HCD­induced increases in blood and liver lipid levels, indicating that DSW may protect against hypercholesterolemia and non­alcoholic hepatic steatosis. In addition, the present study demonstrated that DSW­induced downregulation of lipids in the blood and hepatic lipid accumulation was mediated by enhancement of LDLR expression and suppression of fatty acid synthase and SREBP­1c.

Regulatory mechanism of mineral-balanced deep sea water on hypocholesterolemic effects in HepG2 hepatic cells.

Several previous studies have shown the benefits of deep sea water (DSW) in lipid metabolism. However, the effects of DSW on cellular cholesterol accumulation and synthesis induced by high glucose or free fatty acid plus high glucose [4.5g/L] (FFA/glucose) have not been fully elucidated to date. Herein, we showed the effects of mineral-balanced DSW [magnesium (Mg):calcium (Ca)=3:1] (MB-DSW) on cholesterol metabolism induced by high glucose or FFA/glucose in HepG2 hepatic cells. Moreover, the effects of high ratio Mg DSW [Mg:Ca=40:1] (Mg40) were also investigated. MB-DSW and Mg40 prevented the increase of cellular total cholesterol content in high glucose- or FFA/glucose-treated HepG2 hepatic cells. Furthermore, the inhibition by MB-DSW was closely related to the down-regulation of 3-hydroxy-3-methylglutatryl-CoA reductase (HMGCR) expression and an increase in the AMP-activated protein kinase (AMPK) phosphorylation, leading to decreased cholesterol synthesis in both high glucose- and FFA/glucose-treated conditions. However, this effect was not seen in case of Mg40. In addition, both MB-DSW and Mg40 induced the low-density lipoprotein receptor (LDLR) and diminished the proprotein convertase subtilisin/kexin type 9 (PCSK9) transcriptions in high glucose-treated HepG2 hepatic cells. This result demonstrates that the hypocholesterolemic effects of MB-DSW and Mg40 are mediated with LDL-c clearance through increases of LDLR and its transcription factors, such as peroxisome proliferator-activated receptor-α (PPAR-α), sterol regulatory element-binding protein (SREBP)-1a, and SREBP-2, mRNA synthesis and suppression of PCSK9 transcription. Moreover, apolipoprotein (Apo) A1 transcription was enhanced by MB-DSW and Mg40 without decreasing the expression of Apo B in high glucose-treated HepG2 hepatic cells. However, ApoA1 protein expression was not changed. Taken together, the present investigation suggests that DSW may prevent the high glucose- or FFA/glucose-induced increase of cellular cholesterol levels by inducing LDLR and ApoA1 transcriptions and inhibiting PCSK9 mRNA expression in HepG2 hepatic cells. Additionally, the ratio of Mg in DSW is an important factor that determines whether HMGCR expression and/or AMPK phosphorylation participate in the hypocholesterolemic effects of DSW.

Suppressive effects of a proton beam on tumor growth and lung metastasis through the inhibition of metastatic gene expression in 4T1 orthotopic breast cancer model.

A proton beam is a next generation tool to treat intractable cancer. Although the therapeutic effects of a proton beam are well known, the effect on tumor metastasis is not fully described. Here, we investigated the effects of a proton beam on metastasis in highly invasive 4T1 murine breast cancer cells and their orthotopic breast cancer model. Cells were irradiated with 2, 4, 8 or 16 Gy proton beam, and changes in cell proliferation, survival, and migration were observed by MTT, colony forming and wound healing assays. 4T1 breast cancer cell-implanted BALB/c mice were established and the animals were randomly divided into 4 groups when tumor size reached 200 mm3. Breast tumors were selectively irradiated with 10, 20 or 30 Gy proton beam. Breast tumor sizes were measured twice a week, and breast tumor and lung tissues were pathologically observed. Metastasis-regulating gene expression was assessed with quantitative RT-PCR. A proton beam dose-dependently decreased cell proliferation, survival and migration in 4T1 murine breast cancer cells. Also, growth of breast tumors in the 4T1 orthotopic breast cancer model was significantly suppressed by proton beam irradiation without significant change of body weight. Furthermore, fewer tumor nodules metastasized from breast tumor into lung in mice irradiated with 30 Gy proton beam, but not with 10 and 20 Gy, than in control. We observed correspondingly lower expression levels of urokinase plasminogen activator (uPA), uPA receptor, cyclooxygenase (COX)-2, and vascular endothelial growth factor (VEGF), which are important factors in cancer metastasis, in breast tumor irradiated with 30 Gy proton beam. Proton beam irradiation did not affect expressions of matrix metalloproteinase (MMP)-9 and MMP-2. Taken together, the data suggest that, although proton beam therapy is an effective tool for breast cancer treatment, a suitable dose is necessary to prevent metastasis-linked relapse and poor prognosis.

Crosstalk between Wnt signaling and Phorbol ester-mediated PKC signaling in MCF-7 human breast cancer cells.

Although many studies have implicated the crosstalk between the Wnt and PKC signaling pathways in tumor initiation and progression, the molecular roles of PKC isoforms in the Wnt signaling pathway remain poorly understood. In this study, we explored the contribution of PKC isoforms to canonical and noncanonical Wnt signaling pathway in mediating cell migration and an epithelial-mesenchymal transition (EMT). When MCF-7 cells were treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) for up to 3 weeks, the effect of TPA on Wnt signaling pathway was dramatically different depending on the exposure time. The short term exposure (3 days) of MCF-7 cells to TPA exhibited significant induction of Wnt5a expression, along with the enhanced expression of PKC-α, to promote cell migration, which suggested that activation of noncanonical Wnt signaling pathway is associated with PKC-α. However, the chronic exposure (3 weeks) of cells to TPA completely suppressed Wnt5a expression and the expression of PKC-η and PKC-δ, whereas the expression of Wnt3a and PKC-θ were up-regulated to activate the canonical Wnt signaling pathway. Moreover, the loss of epithelial markers, including E-cadherin and GATA-3, suggested that chronic exposure of TPA stimulates EMT. Taken together, our data suggest that PKC-θ positively regulates the canonical Wnt signaling pathway, and that PKC-η and PKC-δ negatively modulate this signaling pathway.

Lapatinib sensitizes quiescent MDA-MB-231 breast cancer cells to doxorubicin by inhibiting the expression of multidrug resistance-associated protein-1.

The quiescent state plays an important role in tumor recurrence because it protects cancer cells from chemotherapy. Previously, we optimized tumorsphere cultures for in vitro screening methods for targeting quiescent cell population since the majority of cells in tumorspheres are quiescent. In this study, we analyzed efficacies of current chemotherapeutics in tumorsphere assays to seek better strategies for eradicating quiescent cell population. Tumorspheres generated from MDA-MB-231 cells exhibited accumulations of cells in the G0/G1 phase as compared with cells in monolayer culture, suggesting that sphere formation contributes to an increase of quiescent cells. As a result of a decreased doxorubicin uptake, MDA-MB-231 tumorspheres exhibited chemoresistance to both doxorubicin and paclitaxel. Since we found that the enhanced EGFR signaling is characteristics of MDA-MB-231 tumorspheres, the combination effects of chemotherapy with lapatinib, a dual ErbB1/ErbB2 inhibitor, were accessed in tumorsphere assays. Western blot analysis revealed that lapatinib inhibited the phosphorylation of EGFR, AKT and p38 in doxorubicin-treated tumorspheres. The inhibition of EGFR signaling by the treatment with lapatinib suppressed the expression of multidrug resistance-associated protein-1 (MRP-1), leading to increased cytotoxicity of doxorubicin to tumorspheres. Furthermore, blockade of the PI3K/AKT and p38 MAPK signaling pathways resulted in a remarkable decrease in the expression of MRP-1 in doxorubicin-treated tumorspheres. These results demonstrate that lapatinib sensitizes quiescent MDA-MB-231 breast cancer cells to doxorubicin by inhibiting doxorubicin-induced MRP-1 expression via PI3K/AKT and p38 MAPK signaling pathways. Thus, this study suggests that treatment with lapatinib in combination with anti-mitotic drugs maybe a useful approach to improve clinical response by eradicating the quiescent cancer cell population.

Lapatinib enhances the cytotoxic effects of doxorubicin in MCF-7 tumorspheres by inhibiting the drug efflux function of ABC transporters.

Increasing evidences indicate that cancer stem cells are resistant to chemotherapy due to their cell quiescence and the expression of ATP-binding cassette (ABC) transporters. In this study, we utilized tumorsphere cultures to seek better strategies to overcome chemoresistance since tumorsphere cultures have been used widely for the enrichment of cancer stem cells. We found that tumorspheres generated from MCF-7 human breast cancer cells exhibited high proportions of quiescent cells and expressed MDR-1 at elevated levels, leading to resistance to 5-fluorouracil, paclitaxel, and doxorubicin. Because the expression of EGFR/HER2 was increased in MCF-7 tumorspheres, we assessed the combinational effect of the dual ErbB1/ErbB2 inhibitor, lapatinib, with doxorubicin in tumorspheres. The results showed that inhibition of EGFR/HER2 signaling by lapatinib sensitized MCF-7 tumorspheres to doxorubicin by inhibiting the expression of the ABC transporters, MDR-1 and BCRP, and thus, enhancing the intracellular accumulation of doxorubicin. These findings suggest that combinations of lapatinib and cytotoxic anticancer drugs may offer an advantage for treating the drug-resistant cancers.