Cold‐inducible RNA‐binding protein contributes to human antigen R and cyclin E1 deregulation in breast cancer

The cell cycle regulator cyclin E1 is aberrantly expressed in a variety of human cancers. In breast cancer, elevated cyclin E1 correlates with poor outcome, as do high cytoplasmic levels of the stress‐induced RNA‐binding protein human antigen R (HuR). We showed previously that increased cytoplasmic HuR elevates cyclin E1 in MCF‐7 breast cancer cells by stabilizing its mRNA. We show here that cold‐inducible RNA‐binding protein (CIRP) co‐regulates cyclin E1 with HuR in breast cancer cells. CIRP had been shown to interact with HuR in Xenopus laevis oocytes and to be decreased in endometrial cancer. To investigate if human CIRP and HuR co‐regulate cyclin E1, HuR and CIRP levels were altered in MCF‐7 cells and effects on cyclin E1 assessed. Altering HuR expression resulted in a reciprocal change in CIRP expression, while altering CIRP expression resulted in corresponding changes in HuR and cyclin E1 expression. CIRP and HuR co‐precipitated in the presence of RNA and CIRP enhanced HuR binding to the cyclin E1 mRNA and increased cyclin E1 mRNA stability. CIRP co‐localized with HuR predominantly in the nucleus, but also in discrete cytoplasmic foci identified as stress granules (SGs). CIRP overexpression increased the number of HuR‐containing SGs, while its knockdown decreased them. Our results suggest that CIRP positively regulates HuR, ultimately resulting in increased protein synthesis of at least one of its targets. © 2009 Wiley‐Liss, Inc.     

MicroRNA‐34a suppresses the breast cancer stem cell‐like characteristics by downregulating Notch1 pathway

MicroRNAs play pivotal roles in cancer stem cell regulation. Previous studies have shown that microRNA‐34a (miR‐34a) is downregulated in human breast cancer. However, it is unknown whether and how miR‐34a regulates breast cancer stem cells. Notch signaling is one of the most important pathways in stem cell maintenance and function. In this study, we verified that miR‐34a directly and functionally targeted Notch1 in MCF‐7 cells. We reported that miR‐34a negatively regulated cell proliferation, migration, and invasion and breast cancer stem cell propagation by downregulating Notch1. The expression of miR‐34a was negatively correlated with tumor stages, metastasis, and Notch1 expression in breast cancer tissues. Furthermore, overexpression of miR‐34a increased chemosensitivity of breast cancer cells to paclitaxel (PTX) by downregulating the Notch1 pathway. Mammosphere formation and expression of the stemness factor ALDH1 were also reduced in the cells treated with miR‐34a and PTX compared to those treated with PTX alone. Taken together, our results indicate that miR‐34a inhibited breast cancer stemness and increased the chemosensitivity to PTX partially by downregulating the Notch1 pathway, suggesting that miR‐34a/Notch1 play an important role in regulating breast cancer stem cells. Thus miR‐34a is a potential target for prevention and therapy of breast cancer.     

A novel EGR‐1 dependent mechanism for YB‐1 modulation of paclitaxel response in a triple negative breast cancer cell line

Chemotherapy with taxanes such as paclitaxel (PTX) is a key component of triple negative breast cancer (TNBC) treatment. PTX is used in combination with other drugs in both the adjuvant setting and in advanced breast cancer. Because a proportion of patients respond poorly to PTX or relapse after its use, a greater understanding of the mechanisms conferring resistance to PTX is required. One protein shown to be involved in drug resistance is Y‐box binding protein 1 (YB‐1). High levels of YB‐1 have previously been associated with resistance to PTX in TNBCs. In this study, we aimed to determine mechanisms by which YB‐1 confers PTX resistance. We generated isogenic TNBC cell lines that differed by YB‐1 levels and treated these with PTX. Using microarray analysis, we identified EGR1 as a potential target of YB‐1. We found that low EGR1 mRNA levels are associated with poor breast cancer patient prognosis, and that EGR1 and YBX1 mRNA expression was inversely correlated in a TNBC line and in a proportion of TNBC tumours. Reducing the levels of EGR1 caused TNBC cells to become more resistant to PTX. Given that PTX targets cycling cells, we propose a model whereby high YB‐1 levels in some TNBC cells can lead to reduced levels of EGR1, which in turn promotes slow cell cycling and resistance to PTX. Therefore YB‐1 and EGR1 levels are biologically linked and may provide a biomarker for TNBC response to PTX.     

Receptor activator for nuclear factor‐κB ligand signaling promotes progesterone‐mediated estrogen‐induced mammary carcinogenesis

Breast cancer is a leading cause of cancer‐related death in women. Prolonged exposure to the ovarian hormones estrogen and progesterone increases the risk of breast cancer. Although estrogen is known as a primary factor in mammary carcinogenesis, very few studies have investigated the role of progesterone. Receptor activator for nuclear factor‐κB (NF‐κB) ligand (RANKL) plays an important role in progesterone‐induced mammary carcinogenesis. However, the molecular mechanism underlying RANKL‐induced mammary carcinogenesis remains unknown. In our current study, we show that RANKL induces glioma‐associated oncogene homolog 1 (GLI‐1) in estrogen‐induced progesterone‐mediated mammary carcinogenesis. In vivo experiments were carried out using ACI rats and in vitro experiments were carried out in MCF‐7 cells. In ACI rats, mifepristone significantly reduced the incidence of mammary tumors. Likewise, mifepristone also inhibited the proliferation of MCF‐7 cells. Hormone treatments induced RANKL, receptor activator of NF‐κB (RANK), and NF‐κB in a protein kinase B‐dependent manner and inhibited apoptosis by activation of anti‐apoptotic protein Bcl2 in mammary tumors and MCF‐7 cells. Mechanistic studies in MCF‐7 cells reveal that RANKL induced upstream stimulatory factor‐1 and NF‐κB, resulting in subsequent activation of their downstream target GLI‐1. We have identified that progesterone mediates estrogen‐induced mammary carcinogenesis through activation of GLI‐1 in a RANKL‐dependent manner.     

Protein phosphatase 1, regulatory subunit 15B is a survival factor for ERα‐positive breast cancer

Breast cancer is a heterogeneous disease at both the clinical and molecular levels. This heterogeneity may give rise to different therapy responses. Molecular profiling has facilitated identification of signatures for stratifying patients who would potentially benefit from given therapies. Previously, we reported on a subset of genes with the potential for predicting response of primary breast cancer to neoadjuvant chemotherapy. Herein, we report that patients with luminal (estrogen receptor α [ERα]‐expressing) breast cancer were enriched for nonresponders. To identify novel factors that contribute to the survival of breast cancer cells, a loss‐of‐function screen was performed with a subset of genes overexpressed in patients with disease resistant to chemotherapy. This approach led us to identify protein phosphatase 1, regulatory subunit 15B (PPP1R15B) as a factor with a potentially essential role in the survival of ERα‐positive breast cancer cells. Functional analyses showed that PPP1R15B depletion results in impaired proliferation due to unsuccessful transition of cells from G1 to S phase of the cell cycle, and apoptosis induction. Moreover, our data revealed a regulatory role for PPP1R15B in activating ERα. Furthermore, a high level of PPP1R15B mRNA expression was associated with poor outcome following tamoxifen‐based therapy. Accordingly, knockdown of PPP1R15B expression sensitized tamoxifen‐resistant MCF‐7 breast cancer cells to tamoxifen while reducing ERα abundance in these cells. Our findings reveal a novel role for PPP1R15B in the survival and therapy response of ERα‐positive breast cancer and may open new avenues for tumor subtype‐specific therapeutic strategies in the era of personalized medicine.     

Diallyl trisulfide‐induced apoptosis in human cancer cells is linked to checkpoint kinase 1‐mediated mitotic arrest

Growth suppressive effect of diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic, against cultured human cancer cells correlates with checkpoint kinase 1 (Chk1)‐mediated mitotic arrest, but the fate of the cells arrested in mitosis remains elusive. Using LNCaP and HCT‐116 human cancer cells as a model, we now demonstrate that the Chk1‐mediated mitotic arrest resulting from DATS exposure leads to apoptosis. The DATS exposure resulted in G₂ phase and mitotic arrest in both LNCaP and HCT‐116 cell lines. The G₂ arrest was accompanied by downregulation of cyclin‐dependent kinase 1 (Cdk1), cell division cycle (Cdc) 25B, and Cdc25C leading to Tyr15 phosphorylation of Cdk1 (inactivation). The DATS‐mediated mitotic arrest correlated with inactivation of anaphase‐promoting complex/cyclosome as evidenced by accumulation of its substrates cyclinB1 and securin. The DATS treatment increased activating phosphorylation of Chk1 (Ser317) and transient transfection with Chk1‐targeted siRNA conferred significant protection against DATS‐induced mitotic arrest in both cell lines. The Chk1 protein knockdown also afforded partial yet statistically significant protection against apoptotic DNA fragmentation and caspase‐3 activation resulting from DATS exposure in both LNCaP and HCT‐116 cells. Even though DATS treatment resulted in stabilization and Ser15 phosphorylation of p53, the knockdown of p53 protein failed to rescue DATS‐induced mitotic arrest. In conclusion, the results of the present study indicate that Chk1 dependence of DATS‐induced mitotic arrest in human cancer cells is not influenced by the p53 status and cells arrested in mitosis upon DATS exposure are driven to apoptotic DNA fragmentation. © 2009 Wiley‐Liss, Inc.     

miR‐199a‐5p regulates β1 integrin through Ets‐1 to suppress invasion in breast cancer

Increasing evidence has revealed that miR‐199a‐5p is actively involved in tumor invasion and metastasis as well as in the decline of breast cancer tissues. In this research, overexpression of miR‐199a‐5p weakened motility and invasion of breast cancer cells MCF‐7 and MDA‐MB‐231. Upregulation of Ets‐1 increased breast cancer cell invasion, but the mechanism by which miR‐199a‐5p modulates activation of Ets‐1 in breast cancer was not clarified. We investigated the relationship between miR‐199a‐5p and Ets‐1 on the basis of 158 primary breast cancer case specimens, and the results showed that Ets‐1 expression was inversely correlated with endogenous miR‐199a‐5p. Overexpression of miR‐199a‐5p reduced the mRNA and protein levels of Ets‐1 in MCF‐7 and MDA‐MB‐231 cells, whereas anti‐miR‐199a‐5p elevated Ets‐1. siRNA‐mediated Ets‐1 knockdown phenocopied the inhibition invasion of miR‐199a‐5p in vitro. Moreover, luciferase reporter assay revealed that miR‐199a‐5p directly targeted 3′‐UTR of Ets‐1 mRNA. This research revealed that miR‐199a‐5p could descend the levels of β1 integrin by targeting 3′‐UTR of Ets‐1 to alleviate the invasion of breast cancer via FAK/Src/Akt/mTOR signaling pathway. Our results provide insight into the regulation of β1 integrin through miR‐199a‐5p‐mediated Ets‐1 silence and will help in designing new therapeutic strategies to inhibit signal pathways induced by miR‐199a‐5p in breast cancer invasion.     

Overexpression of cyclin D1 enhances taxol induced mitotic death in MCF7 cells

Treatment of MCF7 human breast cancer cells with taxol induces G2/M arrest followed by mitotic death. A moderate overexpression of ectopic cyclin D1 accelerated these G2/M associated events and resulted in a reduced clonogenic survival upon taxol treatment. Taxol treatment resulted in elevated expression of p53 and of p21, which was more pronounced and persistent in cyclin D1 overexpressing cells. Overexpression of cyclin D1 altered sensitivity to taxol by modulating exit from mitosis, which is controlled by p21. These results indicate that overexpression of cyclin D1 sensitizes MCF7 cells to treatment with taxol.     

Inhibition of cyclin E1 overcomes temozolomide resistance in glioblastoma by Mcl‐1 degradation

Glioblastoma (GBM) is one of the major causes of brain cancer‐related mortality worldwide. Temozolomide (TMZ) is an important agent against GBM. Acquired TMZ‐resistance severely limits the chemotherapeutic effect and leads to poor GBM patient survival. To study the underlying mechanism of drug resistance, two TMZ resistant GBM cell lines, A172 and U87, were generated. In this study, the TMZ resistant cells have less apoptosis and cell‐cycle change in response to the TMZ treatment. Western blot results revealed that cyclin E1 was upregulation in TMZ resistant cells. Inhibition or depletion of cyclin E1 re‐sensitized the resistant cells to the TMZ treatment, which indicated the induction of cyclin E1 is the cause of TMZ resistance in GBM cells. Furthermore, we also found the expression of cyclin E1 stabilized the expression of Mcl‐1, which contributes to the TMZ resistance in GBM cells. Finally, our in vivo xenograft data showed that the combination of flavopiridol, a cyclin E1/CDK2 inhibitor, overcomes the TMZ resistant by inducing higher apoptosis. Overall, our data provided a rationale to overcome the TMZ resistant in GBM treatment by inhibiting the cyclin E1 activity.     

Sensitization to TNF‐induced apoptosis by 1,25‐dihydroxy vitamin D3 involves up‐regulation of the TNF receptor 1 and cathepsin B

The active form of vitamin D₃, 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃), induces caspase‐independent apoptosis in MCF‐7 and T47D breast cancer cells. Before the appearance of apoptotic cells at Day 4 after the addition of 1,25(OH)₂D₃, the MCF‐7 cells are sensitized to the caspase‐mediated apoptosis induced by TNF. We studied the mechanism underlying the cross talk between these 2 distinct death pathways in MCF‐7 and T47D cells. Whereas 1,25(OH)₂D₃ pre‐treatment enhanced TNF‐induced apoptosis of TNF sensitive MCF‐7 cells, it failed to render TNF resistant T47D cells sensitive to this cytokine. Opposing to an earlier report suggesting that cytosolic phospholipase A₂ (cPLA₂) mediates the 1,25(OH)₂D₃‐induced sensitization to TNF, we could not detect any cPLA₂ protein in MCF‐7 cells and its overexpression had no effect on cellular sensitivity to 1,25(OH)₂D₃ or the combination with TNF. The sensitization of MCF‐7 cells to TNF‐induced apoptosis by pre‐treatment with 1,25(OH)₂D₃ may instead be partially explained by an increased surface expression of the TNF receptor 1 (TNF‐R1). In line with this, not only the TNF‐induced activation of caspases and apoptosis but also that of NF‐κB was enhanced by 1,25(OH)₂D₃ pre‐treatment. Furthermore, 1,25(OH)₂D₃ enhanced TNF‐induced NF‐κB activation in T47D cells suggesting that it potentiates TNF signaling in general. Interestingly, the lysosomal protease cathepsin B, which expression is up‐regulated by 1,25(OH)₂D₃, was released from the lysosomes upon TNF treatment, and inhibition of its activity rescued 1,25(OH)₂D₃ treated MCF‐7 cells from TNF‐induced apoptosis. In conclusion, 1,25(OH)₂D₃ may enhance TNF‐induced apoptosis by increasing the expression of both the TNF‐R1 and cathepsin B. © 2001 Wiley‐Liss, Inc. © 2001 Wiley‐Liss, Inc.     

Bisphosphonates suppress insulin‐like growth factor 1‐induced angiogenesis via the HIF‐1α/VEGF signaling pathways in human breast cancer cells

Adjunctive chemotherapy with bisphosphonates has been reported to delay bone metastasis and improve overall survival in breast cancer. Aside from its antiresorptive effect, bisphosphonates exhibit antitumor activities, in vitro and in vivo, via several mechanisms, including antiangiogenesis. In this study, we investigated the potential molecular mechanisms underlying the antiangiogenic effect of non–nitrogen‐containing and nitrogen‐containing bisphosphonates, clodronate and pamidronate, respectively, in insulin‐like growth factor (IGF)‐1 responsive human breast cancer cells. We tested whether bisphosphonates had any effects on hypoxia‐inducible factor (HIF)‐1α/vascular endothelial growth factor (VEGF) axis that plays a pivotal role in tumor angiogenesis, and our results showed that both pamidronate and clodronate significantly suppressed IGF‐1‐induced HIF‐1α protein accumulation and VEGF expression in MCF‐7 cells. Mechanistically, we found that either pamidronate or clodronate did not affect mRNA expression of HIF‐1α, but they apparently promoted the degradation of IGF‐1‐induced HIF‐1α protein. Meanwhile, we found that the presence of pamidronate and clodronate led to a dose‐dependent decease in the newly‐synthesized HIF‐1α protein induced by IGF‐1 in breast cancer cells after proteasomal inhibition, thus, indirectly reflecting the inhibition of protein synthesis. In addition, our results indicated that the inhibitory effects of bisphosphonates on the HIF‐1α/VEGF axis are associated with the inhibition of the phosphoinositide 3‐kinase/AKT/mammalian target of rapamycin signaling pathways. Consistently, we demonstrated that pamidronate and clodronate functionally abrogated both in vitro and in vivo tumor angiogenesis induced by IGF‐1‐stimulated MCF‐7 cells. These findings have highlighted an important mechanism of the pharmacological action of bisphosphonates in the inhibition of tumor angiogenesis in breast cancer cells.     

Microtubule‐damaging agents enhance RASSF1A‐induced cell death in lung cancer cell lines

BACKGROUND:

Tumor suppressor gene product RASSF1A has been reported to induce mitotic arrest and apoptosis through its interaction with microtubule and binding to the Ras effector NORE1. Despite this promising antitumor action of microtubule‐targeted drugs, clinical studies demonstrated that paclitaxel (TXL) and vincristine (VCS) have differential antitumor effects, depending on the status of microtubule‐related genes in lung cancer patients. In this study, to provide effective chemotherapeutic treatment for lung cancer patients with the microtubule‐targeted drugs, the authors investigated whether RASSF1A could enhance sensitivity to TXL and VCS, as an intrinsic microtubule modulator, in nonsmall cell lung cancer (NSCLC) cells.

METHODS:

The growth inhibitory effects of TXL and VCS on RASSF1A‐transfected cells were assessed using clonogenic and flow cytometry–based propidium iodide–labeled assay. The levels of mitosis‐related proteins in RASSF1A‐transfected cells after treatment with TXL or VCS were examined by Western blot analysis and in vitro kinase assay.

RESULTS:

RASSF1A enhanced the growth inhibitory effect of TXL and VCS on NSCLC cells and bronchial epithelial transformed cells (BEAS‐2B) by inducing cell cycle arrest at the G₂/M?phase. Accumulation of cyclin B1, G₂/M‐phase–related protein, was observed when RASSF1A‐transfected H1299 cells were treated with TXL or VCS, accompanied with an increase of cyclin A. Inhibition of the activity of cyclin B1/Cdc2 complex by RASSF1A and TXL or VCS was confirmed by kinase assay and knockdown of RASSF1A expression by using small interfering RNA.

CONCLUSIONS:

RSAAF1A protein has a cooperative growth inhibitory effect with microtubule‐targeted drugs through cyclin B1 accumulation on NSCLC cells, suggesting novel insights for the selection of chemotherapeutic agents. Cancer 2009. © 2009 American Cancer Society.     

Critical roles of T‐LAK cell‐originated protein kinase in cytokinesis

We previously reported up‐regulation of T‐LAK cell‐originated protein kinase (TOPK), a novel mitotic kinase, in the great majority of breast cancers. Here we report its critical roles in mitosis, especially in cytokinesis. We found that protein phosphatase 1 alpha (PP1α) inactivation by cyclin‐dependent kinase 1 (CDK1)/cyclin B1 caused enhancement of autophosphorylation of TOPK and resulted in its activation at an early stage of mitosis. Then TOPK interacted with and phosphorylated p97, a member of the AAA+ family of ATPase proteins, through an interaction with p47 protein as an adaptor protein. Interestingly, knockdown of TOPK or p97 in breast cancer cells caused the mitotic failures in the abscission process. This mitotic failure could be rescued by additional exogenous introduction of wild‐type TOPK protein, but not by that of its kinase‐dead form. Our findings suggest that TOPK is indispensable for cancer cell cytokinesis throughout phosphorylation on p97. (Cancer Sci 2009)     

Targeting claudin‐4 enhances chemosensitivity in breast cancer

Triple negative breast cancer (TNBC) is characterized by highly aggressive phenotype, limited treatment options and a poor prognosis. In the present study, we examined the therapeutic effect of anti–claudin (CLDN)‐4 extracellular domain antibody, 4D3, on TNBC. When the expression of CLDN4 and CLDN1 in invasive ductal carcinoma (IDC) was examined in 114 IDC (78 cases from 2004 to 2009 in a single center and 36 cases of tissues array), CLDN1 had lower expression than CLDN4 and was correlated with histological grade. In contrast, expression of CLDN4 was correlated with histological grade, receptor subtype, and stage. CLDN4 expression in human IDC cell lines MCF‐7 (luminal subtype) and MDA‐468 (TNBC) was at the same level. In both cells, paclitaxel (PTX)‐induced growth suppression was enhanced by 4D3. Furthermore, 4D3 increased both intracellular PTX concentration (in both cells) and apoptosis. In the mouse model, 4D3 promoted the antitumor effect of PTX on subcutaneous tumors and reduced lung metastasis. The combination of PTX and 4D3 reduced M2 macrophages and mesenchymal stem cells in the tumor. 4D3 also reduced stemness of the tumors and increased the intratumoral pH. Moreover, concurrent treatment with 4D3, PTX and tamoxifen, or with PTX and tamoxifen in MDA‐468 also showed the same level of antitumor activity and survival as MCF‐7. Furthermore, in a bone metastasis model, combination of PTX and bisphosphonate with 4D3 promoted tumor growth in both cells. Thus, CLDN4 targeting of the antibody facilitated existing therapeutic effects.     

Carcinoembryonic antigen‐related cell adhesion molecule 1 negatively regulates granulocyte colony‐stimulating factor production by breast tumor‐associated macrophages that mediate tumor angiogenesis

Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1), a cell adhesion molecule expressed on epithelial cells and activated immune cells, is downregulated in many cancers and plays a role in inhibition of inflammation in part by inhibition of granulocyte colony‐stimulating factor (G‐CSF) production by myeloid cells. As macrophages are associated with a poor prognosis in breast cancer, but play important roles in normal breast, we hypothesized that CEACAM1 downregulation would lead to tumor promotion under inflammatory conditions. Cocultures of proinflammatory M1 macrophages with CEACAM1 negative MCF7 breast cells produced high levels of G‐CSF (10 ng/mL) compared to CEACAM1‐transfected MCF7/4S cells (1 ng/mL) or anti‐inflammatory M2 macrophage cocultures (0.5 or 0.1 ng/mL, MCF7 or MCF7/4S, respectively). The expression of CEACAM1 on M1s was much greater than for M2s and was observed only in cocultures with either MCF7 or MCF7/4S cells. When M1 macrophages were mixed with MCF7 cells and implanted in murine mammary fat pads of nonobese diabetic/severe combined immunodeficient mice, tumor size and blood vessel density were significantly greater than MCF7 or MCF7/4S only tumors which were hardly detected after 8 weeks of growth. In contrast, M1 cells had a much reduced effect on MCF7/4S tumor growth and blood vessel density, indicating that the tumor inhibitory effect of CEACAM1 is most likely related to its anti‐inflammatory action on inflammatory macrophages. These results support our previous finding that CEACAM1 inhibits both G‐CSF production by myeloid cells and G‐CSF‐stimulated tumor angiogenesis.     

Involvement of Akt/NF‐κB pathway in N6‐isopentenyladenosine‐induced apoptosis in human breast cancer cells

N⁶‐isopentenyladenosine (i6A) inhibits the tumor cell growth by inducing cell apoptosis in various cancer cell lines. However, little is known regarding the mechanisms by which the drug induces cell apoptosis. In this study, we further explored the molecular mechanisms of i6A as an anticancer agent on a human breast cancer cell line MDA MB 231. Treatment with i6A decreased the cell proliferation of MDA MB 231 cells in a dose‐dependent manner by arresting the cells at G₀/G₁ phase. This effect was strongly associated with concomitant decrease in the level of cyclin D1, cyclin E, cdk2, and increase of p21waf1 and p27kip. In addition i6A also induced apoptotic cell death by increasing the expression of Bax, and decreasing the levels of Bcl‐2 and Bcl‐xL, and subsequently triggered mitochondria apoptotic pathway (release of cytochrome c and activation of caspase‐3). We observed that i6A suppressed the nuclear factor kappaB (NF‐κB) pathway and inhibited the Akt activation. The results of this study indicate that i6A decreases cell proliferation and induces apoptotic cell death in human breast cancer cells, possibly by decreasing signal transduction through the Akt/NF‐κB cell survival pathway. © 2010 Wiley‐Liss, Inc.     

Quercetin‐3‐methyl ether inhibits lapatinib‐sensitive and ‐resistant breast cancer cell growth by inducing G2/M arrest and apoptosis

Lapatinib, an oral, small‐molecule, reversible inhibitor of both EGFR and HER2, is highly active in HER2 positive breast cancer as a single agent and in combination with other therapeutics. However, resistance against lapatinib is an unresolved problem in clinical oncology. Recently, interest in the use of natural compounds to prevent or treat cancers has gained increasing interest because of presumed low toxicity. Quercetin‐3‐methyl ether, a naturally occurring compound present in various plants, has potent anticancer activity. Here, we found that quercetin‐3‐methyl ether caused a significant growth inhibition of lapatinib‐sensitive and ‐resistant breast cancer cells. Western blot data showed that quercetin‐3‐methyl ether had no effect on Akt or ERKs signaling in resistant cells. However, quercetin‐3‐methyl ether caused a pronounced G₂/M block mainly through the Chk1‐Cdc25c‐cyclin B1/Cdk1 pathway in lapatinib‐sensitive and ‐resistant cells. In contrast, lapatinib produced an accumulation of cells in the G₁ phase mediated through cyclin D1, but only in lapatinib‐sensitive cells. Moreover, quercetin‐3‐methyl ether induced significant apoptosis, accompanied with increased levels of cleaved caspase 3, caspase 7, and poly(ADP‐ribose) polymerase (PARP) in both cell lines. Overall, these results suggested that quercetin‐3‐methyl ether might be a novel and promising therapeutic agent in lapatinib‐sensitive or ‐resistant breast cancer patients. © 2011 Wiley Periodicals, Inc.