Heregulin induces resistance to lapatinib‐mediated growth inhibition of HER2‐amplified cancer cells

Human epidermal growth factor receptor 2 (HER2) amplification occurs in approximately 20% of gastric and gastroesophageal junction cancers in the United States and European Union. Lapatinib, a dual HER2 and epidermal growth factor receptor tyrosine kinase inhibitor, has demonstrated clinical efficacy in HER2‐amplified cancer cells. However, several studies have shown that some cytokines can mediate resistance to lapatinib using their receptor tyrosine kinase (RTK) pathways. One of these, Heregulin1 (HRG1), can confer resistance to lapatinib‐mediated growth inhibition in HER2‐amplified breast cancer cells, but the underlying mechanisms remain unknown. Here, we investigated whether and how HRG1 causes resistance to lapatinib in gastric and gastroesophageal junction cancers in vitro. HER2‐amplified gastric and gastroesophageal junction cancer cell lines were highly sensitive to lapatinib. Exposure to HRG1 together with lapatinib rescued cells from lapatinib‐induced cell cycle arrest and apoptosis. Downregulation of HER3 with siRNA in the presence of HRG1 re‐sensitized HER2‐amplified cancer cells to lapatinib. Immunoblotting analysis indicated that HRG1 re‐activated HER3 and AKT in the presence of lapatinib, which persisted for at least 72 h. Activation of HER3 and downstream AKT was mediated by residual activity of HER2. HRG1‐mediated resistance could be reduced by PI3K/mTOR inhibitors or by complete inhibition of HER2. Thus, we conclude that HRG1 mediates resistance to lapatinib through HER3 and AKT activation, and that this depends on residual HER2 activity. Lapatinib in combination with anti‐PI3K therapies or more potent HER2 inhibitors would improve the efficacy and avoid the emergence of resistant cells.     

Combined effects of lapatinib and bortezomib in human epidermal receptor 2 (HER2)‐overexpressing breast cancer cells and activity of bortezomib against lapatinib‐resistant breast cancer cells

Lapatinib and bortezomib are highly active against breast cancer cells. Breast cancer patients who initially respond to lapatinib may eventually manifest acquired resistance to this treatment. Thus, the identification of novel agents that may prevent or delay the development of acquired resistance to lapatinib is critical. In the current study, we show that the combination of lapatinib and bortezomib results in a synergistic growth inhibition in human epidermal receptor 2 (HER2)‐overexpressing breast cancer cells and that the combination enhances apoptosis of SK‐BR‐3 cells. Importantly, we found that the combination of lapatinib plus bortezomib more effectively blocked activation of the HER2 pathway in SK‐BR‐3 cells, compared with monotherapy. In addition, we established a model of acquired resistance to lapatinib by chronically challenging SK‐BR‐3 breast cancer cells with increasing concentrations of lapatinib. Here, we showed that bortezomib notably induced apoptosis of lapatinib‐resistant SK‐BR‐3 pools and further inhibited HER2 signaling in the resistant cells. Taken together, the current data indicate a synergistic interaction between lapatinib and bortezomib in HER2‐overexpressing breast cancer cells and provide the rationale for the clinical evaluation of these two noncross‐resistant targeted therapies. The combination of lapatinib and bortezomib may be a potentially novel approach to prevent or delay the onset of acquired resistance to lapatinib in HER2‐overxpressing/estrogen receptor (ER)‐negative breast cancers. (Cancer Sci 2010); 00: 000–000     

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.     

Antitumor effects of novel compound,guttiferone K,on colon cancer by p21Waf1/Cip1‐mediated G0/G1 cell cycle arrest and apoptosis

Low selectivity is one of the major problems of currently used anticancer drugs, therefore, there is a high demand for novel, selective antitumor agents. In this study, the anticancer effects and mechanisms of guttiferone K (GUTK), a novel polyprenylated acylphloroglucinol derivative isolated from Garcinia cowa Roxb., were examined for its development as a novel drug targeting colon cancer. GUTK concentration‐ and time‐dependently reduced the viability of human colon cancer HT‐29 cells (IC₅₀ value 5.39 ± 0.22 μM) without affecting the viability of normal human colon epithelial CCD 841 CoN cells and induced G₀/G₁ cell cycle arrest in HT‐29 cells by down‐regulating cyclins D1, D3 and cyclin‐dependent kinases 4 and 6, while selectively restoring p21Waf1/Cip1 and p27Kip1 to levels comparable to those observed in normal colon cells, without affecting their levels in normal cells. GUTK (10.0 μM) induced cleavage of PARP, caspases‐3, ‐8 and ‐9 and chromatin condensation to stimulate caspase‐3‐mediated apoptosis. The addition of a JNK inhibitor, SP600125, partially reversed GUTK‐induced caspase‐3 activity, indicating the possible involvement of JNK in GUTK‐induced apoptosis. Furthermore, GUTK (10 mg/kg, i.p.) significantly decreased the tumor volume in a syngeneic colon tumor model when used alone or in combination with 5‐fluorouracil without toxicity to the mice. Immunohistochemical staining of the tumor sections revealed a mechanism involving an increase in cleaved caspase‐3 and a decrease in cell proliferation marker Ki‐67. Our results support GUTK as a promising novel, potent and selective antitumor drug candidate for colon cancer.     

Anti‐proliferative and apoptosis‐inducing activity of lycopene against three subtypes of human breast cancer cell lines

Although lycopene, a major carotenoid component of tomatoes, has been suggested to attenuate the risk of breast cancer, the underlying preventive mechanism remains to be determined. Moreover, it is not known whether there are any differences in lycopene activity among different subtypes of human breast cancer cells. Using ER/PR positive MCF‐7, HER2‐positive SK‐BR‐3 and triple‐negative MDA‐MB‐468 cell lines, we investigated the cellular and molecular mechanism of the anticancer activity of lycopene. Lycopene treatment for 168 consecutive hours exhibited a time‐dependent and dose‐dependent anti‐proliferative activity against these cell lines by arresting the cell cycle at the G₀/G₁ phase at physiologically achievable concentrations found in human plasma. The greatest growth inhibition was observed in MDA‐MB‐468 where the sub‐G₀/G₁ apoptotic population was significantly increased, with demonstrable cleavage of PARP. Lycopene induced strong and sustained activation of the ERK1/2, with concomitant cyclin D1 suppression and p21 upregulation in these three cell lines. In triple negative cells, lycopene inhibited the phosphorylation of Akt and its downstream molecule mTOR, followed by subsequent upregulation of proapoptotic Bax without affecting anti‐apoptotic Bcl‐xL. Taken together, these data indicate that the predominant anticancer activity of lycopene in MDA‐MB‐468 cells suggests a potential role of lycopene for the prevention of triple negative breast cancer.     

Expression of CD24 is associated with HER2 expression and supports HER2‐Akt signaling in HER2‐positive breast cancer cells

Human epidermal growth factor receptor 2 (HER2)‐positive breast cancer is treated with HER2‐targeted agents, such as trastuzumab and lapatinib, that suppress signaling by phosphatidylinositol 3‐kinase (PI3K)‐Akt and MAPK pathways. However, resistance to HER2‐targeted therapy remains a major clinical problem. Overexpression of CD24 has been detected in many cancers and is associated with a poor prognosis in women with breast cancer. HER2‐positive breast tumors are predominantly positive for CD24, suggesting that the expression of the two molecules is related. To investigate the relation between HER2 and CD24, we overexpressed HER2 in breast cancer cells that were triple‐negative for the estrogen receptor, progesterone receptor and HER2. We found that expression of CD24 was increased by stable overexpression of HER2. Flow cytometry thus revealed that the percentage of CD24‐positive cells was markedly higher in the HER2‐positive fraction than in the HER2‐negative fraction. Knockdown of CD24 in breast cancer cells positive for endogenous HER2 downregulated HER2 expression, whereas knockdown of HER2 did not affect the expression of CD24. Knockdown of CD24 also suppressed the phosphorylation of Akt, which functions downstream of HER2 and PI3K to promote cell survival. Moreover, knockdown of CD24 increased the sensitivity of HER2‐positive breast cancer cells to lapatinib treatment. Our results thus indicate that CD24 supports both the expression of HER2 and the consequent activation of PI3K‐Akt signaling. Furthermore, CD24 may contribute to resistance to HER2‐targeted therapy and, therefore, is itself a potential therapeutic target in HER2‐positive breast cancer.     

5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone‐induced cell death in human Leukemia cells is dependent on caspases and activates the MAPK pathway

Flavonoids are polyphenolic compounds which display a vast array of biological activities and are promising anticancer agents. In this study we investigated the effect of 5,7,3′‐trihydroxy‐3,4′‐dimethoxyflavone (THDF) on viability of nine human tumor cell lines and found that it was highly cytotoxic against leukemia cells. THDF induced G₂–M phase cell‐cycle arrest and apoptosis through a caspase‐dependent mechanism involving cytochrome c release, processing of multiple caspases (caspase‐3, ‐6, ‐7, and ‐9) and cleavage of poly(ADP‐ribose) polymerase. Overexpression of the protective mitochondrial proteins Bcl‐2 and Bcl‐x_L conferred partial resistance to THDF‐induced apoptosis. This flavonoid induced the phosphorylation of members of the mitogen‐activated protein kinases (MAPKs) family and cell death was attenuated by inhibition of c‐jun N‐terminal kinases/stress‐activated protein kinases (JNK/SAPK) and of extracellular signal‐regulated kinases (ERK) 1/2. In the present study we report that THDF‐induced cell death is mediated by an intrinsic dependent apoptotic event involving mitochondria and MAPKs, and through a mechanism independent of the generation of reactive oxygen species. The results suggest that THDF could be useful in the development of novel anticancer agents. Mol. Carcinog. © 2010 Wiley‐Liss, Inc.     

PAI‐1 induces Src inhibitor resistance via CCL5 in HER2‐positive breast cancer cells

Tyrosine kinase Src is overexpressed and activated in various tumors, including breast cancer, and is supposed to promote cancer formation and development. Src inhibitors have been developed recently and have shown efficacy in breast cancer as a single agent or in combination with anti‐HER2 antibodies or chemotherapy. Unfortunately, the potency of Src inhibitor is limited by the development of drug resistance. In our study, we established an Src inhibitor saracatinib‐resistant breast cancer cell line (SKBR‐3/SI) for the first time and by evaluating mRNA expression profile, we found that plasminogen activator inhibitor‐1 (PAI‐1) was upregulated in saracatinib‐resistant cells compared to the parent cells. Further study demonstrated that PAI‐1 might induce saracatinib resistance in breast cancer cells by increasing the secretion of chemokine (C‐C motif) ligand 5 (CCL5). Functional assays showed that PAI‐1 and CCL5 overexpression promoted cell proliferation and migration in breast cancer cells, while inhibition of PAI‐1 and CCL5 decreased cell proliferation and migration in saracatinib‐resistant cells. We also showed that targeting PAI‐1 or CCL5 could reverse saracatinib resistance, which deserves more attention in clinical settings.     

Aberrant intracellular metabolism of T‐DM1 confers T‐DM1 resistance in human epidermal growth factor receptor 2‐positive gastric cancer cells

Trastuzumab emtansine (T‐DM1), an antibody–drug conjugate (ADC) consisting of human epidermal growth factor receptor 2 (HER2)‐targeted mAb trastuzumab linked to antimicrotubule agent mertansine (DM1), has been approved for the treatment of HER2‐positive metastatic breast cancer. Acquired resistance has been a major obstacle to T‐DM1 treatment, and mechanisms remain incompletely understood. In the present study, we established a T‐DM1‐resistant N87‐KR cell line from HER2‐positive N87 gastric cancer cells to investigate mechanisms of acquired resistance and develop strategies for overcoming it. Although the kinetics of binding, internalization, and externalization of T‐DM1 were the same in N87‐KR cells and N87 cells, N87‐KR was strongly resistant to T‐DM1, but remained sensitive to both trastuzumab and DM1. T‐DM1 failed to inhibit microtubule polymerization in N87‐KR cells. Consistently, lysine‐MCC‐DM1, the active T‐DM1 metabolite that inhibits microtubule polymerization, accumulated much less in N87‐KR cells than in N87 cells. Furthermore, lysosome acidification, achieved by vacuolar H⁺‐ATPase (V‐ATPase), was much diminished in N87‐KR cells. Notably, treatment of sensitive N87 cells with the V‐ATPase selective inhibitor bafilomycin A1 induced T‐DM1 resistance, suggesting that aberrant V‐ATPase activity decreases T‐DM1 metabolism, leading to T‐DM1 resistance in N87‐KR cells. Interestingly, HER2‐targeted ADCs containing a protease‐cleavable linker, such as hertuzumab‐vc‐monomethyl auristatin E, were capable of efficiently overcoming this resistance. Our results show for the first time that a decrease in T‐DM1 metabolites induced by aberrant V‐ATPase activity contributes to T‐DM1 resistance, which could be overcome by HER2‐targeted ADCs containing different linkers, including a protease‐cleavable linker. Accordingly, we propose that V‐ATPase activity in lysosomes is a novel biomarker for predicting T‐DM1 resistance.     

PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro

Introduction

Alterations in cell cycle regulators have been implicated in human malignancies including breast cancer. PD 0332991 is an orally active, highly selective inhibitor of the cyclin D kinases (CDK)4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. To identify predictors of response, we determined the in vitro sensitivity to PD 0332991 across a panel of molecularly characterized human breast cancer cell lines.

Methods

Forty-seven human breast cancer and immortalized cell lines representing the known molecular subgroups of breast cancer were treated with PD 0332991 to determine IC₅₀ values. These data were analyzed against baseline gene expression data to identify genes associated with PD 0332991 response.

Results

Cell lines representing luminal estrogen receptor-positive (ER+) subtype (including those that are HER2 amplified) were most sensitive to growth inhibition by PD 0332991 while nonluminal/basal subtypes were most resistant. Analysis of variance identified 450 differentially expressed genes between sensitive and resistant cells. pRb and cyclin D₁ were elevated and CDKN2A (p16) was decreased in the most sensitive lines. Cell cycle analysis showed G₀/G₁ arrest in sensitive cell lines and Western blot analysis demonstrated that Rb phosphorylation is blocked in sensitive lines but not resistant lines. PD 0332991 was synergistic with tamoxifen and trastuzumab in ER+ and HER2-amplified cell lines, respectively. PD 0332991 enhanced sensitivity to tamoxifen in cell lines with conditioned resistance to ER blockade.

Conclusions

These studies suggest a role for CDK4/6 inhibition in some breast cancers and identify criteria for patient selection in clinical studies of PD 0332991.     

Neoadjuvant Dual HER2‐Targeted Therapy With Lapatinib and Trastuzumab Improves Pathologic Complete Response in Patients With Early Stage HER2‐Positive Breast Cancer: A Meta‐Analysis of Randomized Prospective Clinical Trials

Background.

Randomized clinical trials (RCT) that evaluated the addition of lapatinib to trastuzumab plus neoadjuvant chemotherapy (NAC) in patients with HER2-positive, operable breast cancer revealed a questionable improvement in pathologic complete response (pCR) rate. We performed a meta-analysis of prospective RCTs that examined the effect of adding lapatinib to trastuzumab and NAC on pCR rate.

Methods.

PubMed databases and abstracts from the proceedings of the American Society of Clinical Oncology and the San Antonio Breast Cancer Symposium were searched for RCTs that compared lapatinib plus trastuzumab and NAC with trastuzumab in combination with NAC and that included pCR as the primary outcome. Our main objective was to estimate the effect of adding lapatinib to trastuzumab plus NAC on pCR rate, defined as no residual invasive cancer in breast and axillary lymph nodes.

Results.

In total, 1,017 patients with early stage breast cancer from 5 trials were included. Four trials examined the addition of lapatinib to trastuzumab plus NAC; this resulted in statistically significant improvement in pCR, defined as no residual carcinoma in breast and lymph nodes. The pCR rate was 55.76% and 38.36% in the lapatinib plus trastuzumab and the trastuzumab plus NAC arms, respectively (odds ratio [OR]: 1.94; 95% confidence interval [CI]: 1.44–2.60). In three trials, the rates of pCR, defined as no residual invasive carcinoma in breast only, for the lapatinib plus trastuzumab and trastuzumab-alone groups were 55.01% and 40.70%, respectively, also resulting in significant improvement (OR: 1.78; 95% CI: 1.27–2.50).

Conclusion.

The addition of lapatinib to trastuzumab in combination with neoadjuvant chemotherapy significantly improves pCR rates in patients with HER2-positive breast cancer.     

Tissue inhibitor of metalloproteinase‐1 protects MCF‐7 breast cancer cells from paclitaxel‐induced apoptosis by decreasing the stability of cyclin B1

Paclitaxel (PTX) is a very effective drug in treating tumors. It disturbs microtubule dynamics and impairs the transition of cells from metaphase to anaphase in mitosis, leading to cell death by apoptosis. However, the effectiveness of PTX in cancer chemotherapy is hampered by drug resistance in some patients. Tissue inhibitor of metalloproteinase‐1 (TIMP‐1) is well known to be capable of inhibiting apoptosis. Elevated tumor tissue TIMP‐1 levels have been significantly associated with a poor response to chemotherapy. We hypothesized that TIMP‐1 could reduce the sensitivity of breast cancer cells to PTX by inhibiting apoptosis. To test this hypothesis, we first examined the effects of TIMP‐1 on the apoptosis induced by PTX and investigated the effects of TIMP‐1 on the expression and stability of cyclin B1 that critically regulates the metaphase to anaphase transition during mitosis in MCF‐7 breast cancer cells. Our data demonstrate that TIMP‐1 could significantly decrease the sensitivity of MCF‐7 cells to PTX‐induced apoptosis, attenuate mitotic blockage in G₂/M, and enhance the degradation of cyclin B1. To further investigate whether the inhibitory effect of TIMP‐1 on PTX‐induced apoptosis is mediated by lowering levels of cyclin B1, a cyclin B1‐expression plasmid was transfected into clone overexpressing TIMP‐1. The levels of PTX‐induced apoptosis were then analyzed. The data showed that the TIMP‐1‐based decrease in PTX‐induced apoptosis was reversed by cyclin B1. Our data indicate that TIMP‐1 can protect breast cancer cells from PTX‐induced apoptosis by decreasing the stability of cyclin B1.     

Combining lapatinib and pertuzumab to overcome lapatinib resistance due to NRG1-mediated signalling in HER2-amplified breast cancer

Acquired resistance to lapatinib, an inhibitor of EGFR and HER2 kinases, is common. We found that reactivation of EGFR, HER2 and HER3 occurred within 24 hours of lapatinib treatment after their initial dephosphorylation. This was associated with increased expression of NRG1 in cells treated with lapatinib. Exogenous NRG1 partially rescued breast cancer cells from growth inhibition by lapatinib. In addition, both parental and lapatinib-resistant breast cancer cells were sensitive to SGP1, which inhibits binding of NRG1 and other HER3 ligands. Addition of pertuzumab to lapatinib further inhibited NRG1-induced signalling, which was not fully inhibited by either drug alone. In animal model, a combination of pertuzumab to lapatinib induced a greater tumor regression than either lapatinib or pertuzumab monotherapy. This novel combination treatment may provide a promising strategy in clinical HER2-targeted therapy and may inhibit a subset of lapatinib-resistant breast cancer, although the group of patients that will respond to this therapy requires further stratification.     

DS‐8201a,a new HER2‐targeting antibody–drug conjugate incorporating a novel DNA topoisomerase I inhibitor,overcomes HER2‐positive gastric cancer T‐DM1 resistance

Anti‐HER2 therapies are beneficial for patients with HER2‐positive breast or gastric cancer. T‐DM1 is a HER2‐targeting antibody–drug conjugate (ADC) comprising the antibody trastuzumab, a linker, and the tubulin inhibitor DM1. Although effective in treating advanced breast cancer, all patients eventually develop T‐DM1 resistance. DS‐8201a is a new ADC incorporating an anti‐HER2 antibody, a newly developed, enzymatically cleavable peptide linker, and a novel, potent, exatecan‐derivative topoisomerase I inhibitor (DXd). DS‐8201a has a drug‐to‐antibody‐ratio (DAR) of 8, which is higher than that of T‐DM1 (3.5). Owing to these unique characteristics and unlike T‐DM1, DS‐8201a is effective against cancers with low‐HER2 expression. In the present work, T‐DM1‐resistant cells (N87‐TDMR), established using the HER2‐positive gastric cancer line NCI‐N87 and continuous T‐DM1 exposure, were shown to be susceptible to DS‐8201a. The ATP‐binding cassette (ABC) transporters ABCC2 and ABCG2 were upregulated in N87‐TDMR cells, but HER2 overexpression was retained. Furthermore, inhibition of ABCC2 and ABCG2 by MK571 restored T‐DM1 sensitivity. Therefore, resistance to T‐DM1 is caused by efflux of its payload DM1, due to aberrant expression of ABC transporters. In contrast to DM1, DXd payload of DS‐8201a inhibited the growth of N87‐TDMR cells in vitro. This suggests that either DXd may be a poor substrate of ABCC2 and ABCG2 in comparison to DM1, or the high DAR of DS‐8201a relative to T‐DM1 compensates for increased efflux. Notably, N87‐TDMR xenograft tumor growth was prevented by DS‐8201a. In conclusion, the efficacy of DS‐8201a as a treatment for patients with T‐DM1‐resistant breast or gastric cancer merits investigation.     

Preclinical antitumor activity of S‐222611, an oral reversible tyrosine kinase inhibitor of epidermal growth factor receptor and human epidermal growth factor receptor 2

Epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) are validated molecular targets in cancer therapy. Dual blockade has been explored and one such agent, lapatinib, is in clinical practice but with modest activity. Through chemical screening, we discovered a novel EGFR and HER2 inhibitor, S‐222611, that selectively inhibited both kinases with IC₅₀s below 10 nmol/L. S‐222611 also inhibited intracellular kinase activity and the growth of EGFR‐expressing and HER2‐expressing cancer cells. In addition, S‐222611 showed potent antitumor activity over lapatinib in a variety of xenograft models. In evaluations with two patient‐oriented models, the intrafemoral implantation model and the intracranial implantation model, S‐222611 exhibited excellent activity and could be effective against bone and brain metastasis. Compared to neratinib and afatinib, irreversible EGFR/HER2 inhibitors, S‐222611 showed equivalent or slightly weaker antitumor activity but a safer profile. These results indicated that S‐222611 is a potent EGFR and HER2 inhibitor with substantially better antitumor activity than lapatinib at clinically relevant doses. Considering the safer profile than for irreversible inhibitors, S‐222611 could be an important option in future cancer therapy.     

Betuletol 3‐methyl ether induces G2‐M phase arrest and activates the sphingomyelin and MAPK pathways in human leukemia cells

Betuletol 3‐methyl ether (BME) is a natural phenylbenzo‐γ‐pyrone that inhibits cell proliferation in human tumor cell lines and induces apoptotic cell death in HL‐60 cells. Here we show that BME displays strong cytotoxic properties in several human leukemia cell lines (U937, K‐562, THP‐1, Jurkat, and Molt‐3) and in cells that over‐express two anti‐apoptotic proteins, namely Bcl‐2 and Bcl‐x_L. BME arrested HL‐60 cells at G₂‐M phase of the cell cycle, which was associated with the accumulation of cyclin B1 and p21^Cip1. Fluorescence microscopy experiments suggest that BME blocked the cell cycle in mitosis. The in vivo tubulin polymerization assay shows that BME inhibits tubulin polymerization and causes similar changes of cellular microtubule network as colchicine. Our results demonstrate that BME‐induced cell death is (i) triggered in human myeloid leukemia cell that over‐express Bcl‐2 and Bcl‐x_L, and (ii) associated with loss of inner mitochondrial membrane potential (ΔΨm) and an increase in reactive oxygen species (ROS). Although ROS increased in response to BME, this did not seem to play a pivotal role in the apoptotic process since the anti‐oxidant trolox was unable to provide cell protection. The treatment of HL‐60 cells with BME induces the activation of mitogen‐activated protein kinases (MAPKs) such as c‐Jun N‐terminal kinases, p38 mitogen‐activated protein kinases and extracellular signal‐regulated kinases (ERK)1/2 and stimulates the acid sphingomyelinase with concomitant ceramide generation. The findings of this study suggest that BME could be useful in the development of novel anticancer agents. © 2009 Wiley‐Liss, Inc.     

The dual EGFR/HER‐2 tyrosine kinase inhibitor lapatinib sensitizes colon and gastric cancer cells to the irinotecan active metabolite SN‐38

Members of the human epidermal receptor (HER) family are frequently associated with aggressive disease and poor prognosis in multiple malignancies. Lapatinib is a dual tyrosine kinase inhibitor targeting the epidermal growth factor receptor (EGFR) and HER‐2. This study evaluated the therapeutic potential of lapatinib, alone and in combination with SN‐38, the active metabolite of irinotecan (CPT‐11), in colon and gastric cancer cell lines. Concentration‐dependent antiproliferative effects of both lapatinib and SN‐38 were observed in all colon and gastric cancer cell lines tested but varied significantly between individual cell lines (lapatinib range 0.08–11.7 μM; SN‐38 range 3.6–256 nM). Lapatinib potently inhibited the growth of a HER‐2 overexpressing gastric cancer cell line and demonstrated moderate activity in gastric and colon cancer cells with detectable HER‐2 expression. The combination of lapatinib and SN‐38 interacted synergistically to inhibit cell proliferation in all colon and gastric cancer cell lines tested. Cotreatment with lapatinib and SN‐38 also resulted in enhanced cell cycle arrest and the induction of apoptosis with subsequent cellular pharmacokinetic analysis demonstrating that lapatinib promoted the increased intracellular accumulation and retention of SN‐38 when compared to SN‐38 treatment alone. Finally, the combination of lapatinib and CPT‐11 demonstrated synergistic antitumor efficacy in the LoVo colon cancer mouse xenograft model with no apparent increase in toxicity compared to CPT‐11 monotherapy. These results provide compelling preclinical rationale indicating lapatinib to be a potentially efficacious chemotherapeutic combination partner for irinotecan in the treatment of gastrointestinal carcinomas. © 2009 UICC