Evaluation of uptake,cytotoxicity and inflammatory effects in respiratory cells exposed to pristine and ‐OH and ‐COOH functionalized multi‐wall carbon nanotubes

Toxic effects were reported for pristine‐multi‐wall carbon nanotubes (p‐MWCNTs) while the role of the functionalization on MWCNT‐induced toxicity is not yet well defined. We evaluated on human alveolar (A549) epithelial cells and normal bronchial (BEAS‐2B) cells exposed to p‐MWCNTs, MWCNTs‐OH and MWCNTs‐COOH: uptake by TEM, cell viability by different assays, membrane damage by the LDH assay and cytokine release by ELISA. The aims of the present study were to: (i) confirm MWCNT cytotoxicity mechanisms hypothesized in our previous studies; (ii) identify the most reliable viability assay to screen MWCNT toxicity; and (iii) to test our model to clarify the role of functionalization on MWCNT‐induced toxicity. In A549 cells, p‐MWCNTs and MWCNTs‐OH were localized free in the cytoplasm and inside vacuoles whereas MWCNTs‐COOH were confined inside filled cytoplasmic vesicles. WST‐1 and Trypan blue assays showed in A549 cells a similar slight viability reduction for all MWCNTs whereas in BEAS‐2B cells WST1 showed a high viability reduction at the highest concentrations, particularly for MWCNTs‐COOH. The MTT assay showed a false cytotoxicity as a result of MWCNTs‐interference. Pristine and MWCNTs‐COOH induced membrane damage, particularly in BEAS‐2B cells. MWCNTs‐COOH induced interleukin‐6 (IL‐6) and IL‐8 release in A549 cells whereas p‐MWCNTs induced IL‐8 release in BEAS‐2B cells. MWCNTs intracellular localization in A549 cells confirms the toxicity mechanisms previously hypothesized, with p‐MWCNTs disrupting the membrane and vesicle‐confined MWCNTs‐COOH inducing inflammation. WST‐1 was more reliable than MTT to test MWCNT‐toxicity. BEAS‐2B cells were more susceptible then A549 cells, particularly to MWCNT‐COOH cytotoxicity. Our results confirm the toxicity of p‐MWCNTs and demonstrate, also for the two kinds of tested functionalized MWCNTs toxic effects with a different mechanism of action. Copyright © 2015 John Wiley & Sons, Ltd.     

表没食子儿茶素没食子酸酯联合曲妥珠单抗对HER2过表达乳腺癌细胞增殖的影响及其机制

目的 研究表没食子儿茶素没食子酸酯（EGCG）联合曲妥珠单抗对人表皮生长因子受体2(HER2)过表达乳腺癌细胞增殖的影响及其作用机制。方法 表达纯化曲妥珠单抗;用CCK-8细胞增殖检测试剂盒（CCK8）检测不同浓度EGCG、曲妥珠单抗及两药联用对HER2过表达乳腺癌细胞BT474、SK-BR-3的增殖抑制作用;用Western blot法检测EGCG、曲妥珠单抗及两药联用对BT474乳腺癌细胞中HER2,表皮生长因子受体（EGFR）,丝裂原激活的蛋白激酶（MAPK）和蛋白激酶B(Akt)及它们的磷酸化蛋白的表达水平的影响。结果 细胞增殖试验结果显示,EGCG、曲妥珠单抗以及二者联用均能有效抑制BT474和SK-BR-3细胞的增殖,且在一定浓度范围内,EGCG与曲妥珠单抗联用显示出协同增殖抑制作用。Western blot结果显示EGCG、曲妥珠单抗以及二者联合均能抑制BT474细胞中Akt,MAPK,EGFR,HER2的磷酸化蛋白表达,与单药相比,二者联合抑制作用显著增强,其差异具有统计学意义(P<0.05)。结论 EGCG联合曲妥珠单抗能协同抑制HER2过表达乳腺癌细胞的增殖,...     

Preparation and preclinical evaluation of 131I‐trastuzumab for breast cancer

Trastuzumab that targets the human epidermal growth factor receptor type 2 (HER2) is known to benefit patients with HER2+ metastatic breast cancer. The objective was to explore the potential of ¹³¹I‐trastuzumab for treatment of breast cancers. Radioiodination of trastuzumab was carried out by chloramine‐T method, purified by using PD‐10 column, and characterized by size exclusion high‐performance liquid chromatography on a gel column. In vitro studies were carried out in HER2+ cells to determine the specificity of the radioimmunoconjugate. Uptake and retention of ¹³¹I‐trastuzumab were determined by biodistribution studies in tumor‐bearing non‐obese diabetic/severe combined immunodeficiency and normal severe combined immunodeficiency mice. The radiochemical purity (RCP) of ¹³¹I‐trastuzumab was 98 ± 0.4% with retention time of 17 minutes by high‐performance liquid chromatography. In vitro stability studies exhibited RCP of more than 90% in serum at 37°C after 120 hours of radioiodination. In vitro cell binding with ¹³¹I‐trastuzumab in HER2+ cells showed binding of 28% to 35% which was inhibited significantly, with unlabeled trastuzumab confirming its specificity. K_d value of ¹³¹I‐trastuzumab was 0.5 nM, while its immunoreactivity was more than 80%. Uptake of more than 12% and retention were observed in the tumors up to 120 hours p.i. ¹³¹I‐trastuzumab prepared in‐house‐exhibited RCP of more than 98%, excellent immunoreactivity, affinity to HER2+ cell lines and good tumor uptake thereby indicating its potential for further evaluation in HER2+ breast cancers.     

Trastuzumab,a humanized anti-HER2 monoclonal antibody,for the treatment of breast cancer

The HER2/neu gene encodes a 185 kDa transmembrane receptor (HER2) that belongs to the epidermal growth factor receptor family and has intrinsic tyrosine kinase activity. HER2 is overexpressed in 25-30% of breast cancers and is suggested to have a direct role in the pathogenesis and clinical aggressiveness of HER2 overexpressing tumors. A murine monoclonal antibody, 4D5, directed against the extracellular domain of HER2, is a potent inhibitor of growth of human breast cancer cells overexpressing HER2 in vitro and in xenograft models. To facilitate clinical investigation, 4D5 was humanized by inserting the complementary determining regions of 4D5 into the framework of a consensus human IgG1. The resulting recombinant humanized anti-HER2 MAb, trastuzumab, was found to inhibit the growth of human cancer cells and tumor xenografts overexpressing HER2. Data from phase II trials in women with breast cancer whose tumors overexpress HER2 have shown that trastuzumab has a favorable toxicity profile, is active as a single agent and induces long-lasting objective tumor responses. In combination studies, there was no evidence that trastuzumab enhanced the toxicity of cisplatin and the pharmacokinetic parameters of trastuzumab were unaltered by coadministration of cisplatin. Furthermore, clinical response rates were higher than those reported with either agent alone in a similar patient population. Results of a multicenter, phase III clinical trial of chemotherapy (doxorubicin- or paclitaxel-based) plus trastuzumab as compared to chemotherapy alone in patients with advanced breast cancers overexpressing HER2 showed a significant enhancement in the effects of chemotherapy on time to disease progression, response rates and survival with coadministration of trastuzumab, without increases in overall severe adverse events. Myocardial dysfunction syndrome, similar to that observed with anthracyclines, was reported more commonly with chemotherapy plus trastuzumab. Positive results from clinical studies led to the approval of trastuzumab in the U.S in October 1998 for the treatment of metastatic breast cancer in patients with tumors overexpressing HER2. Since then, the MAb has also been marketed in Switzerland and Canada.     

Trastuzumab: a review of its use in the treatment of metastatic breast cancer overexpressing HER2.

Trastuzumab is a humanised monoclonal antibody developed to target the HER2 receptor which is overexpressed by some cancer cells, including 25 to 30% of breast cancers. Binding with high affinity to the extracellular domain of HER2, trastuzumab inhibits the proliferation of tumour cells that overexpress HER2. A large well designed multicentre study found that the addition of trastuzumab to either an anthracycline plus cyclophosphamide or to paclitaxel, as first-line therapy for metastatic breast cancer overexpressing the HER2 receptor, significantly increased time to disease progression, rate of objective response, duration of response and survival compared with chemotherapy alone. Single-agent trastuzumab was associated with an objective response in 15% of extensively pretreated patients with metastatic breast cancer overexpressing HER2, and 26% of previously untreated patients. Patients with a HER2 overexpression level of 3+ using immunohistochemical (IHC) assay or a positive HER2 result using fluorescence in situ hybridisation (FISH), benefit more from trastuzumab therapy than those with tumours overexpressing at a level of 2+. Trastuzumab has demonstrated synergistic action with several chemotherapy agents preclinically but the optimal combination clinically is yet to be determined. Trastuzumab is generally well tolerated by most patients; the most significant adverse effects being acute fever and/or chills and the potential to cause cardiac dysfunction. Serious adverse events, including anaphylaxis and death, have occurred in 0.25% of patients. Symptomatic or asymptomatic cardiac dysfunction occurred in 27% of patients receiving an anthracycline and cyclophosphamide combined with trastuzumab. Thus, combination therapy with anthracyclines is not recommended. Symptomatic or asymptomatic cardiac dysfunction occurred in 13% of patients receiving trastuzumab plus paclitaxel and in 4.7% of patients receiving trastuzumab alone. CONCLUSION: Intravenous trastuzumab is effective as a single-agent, and in combination with chemotherapy it significantly improves the median time to disease progression and survival time in patients with metastatic breast cancer overexpressing the HER2 receptor compared with chemotherapy alone. Cardiotoxicity is the main concern with therapy; particularly in patients with pre-existing cardiac dysfunction, the elderly and in combination with, or following, anthracyclines. Trastuzumab is indicated for use with paclitaxel as first-line therapy or as a single agent in second- or third-line treatment regimens for patients with metastatic breast cancer overexpressing HER2. Investigation is ongoing to ascertain the optimal combination regimen containing trastuzumab and antineoplastic agents. In addition, current research is focusing on the optimal timing, sequencing and duration of therapy as well as administration in the neoadjuvant and adjuvant setting.     

[64Cu]‐labelled trastuzumab: optimisation of labelling by DOTA and NODAGA conjugation and initial evaluation in mice

The human epidermal growth factor receptor‐2 (HER2) is overexpressed in 20–30% of all breast cancer cases, leading to increased cell proliferation, growth and migration. The monoclonal antibody, trastuzumab, binds to HER2 and is used for treatment of HER2‐positive breast cancer. Trastuzumab has previously been labelled with copper‐64 by conjugation of a 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) chelator. The aim of this study was to optimise the ⁶⁴Cu‐labelling of DOTA‐trastuzumab and as the first to produce and compare with its 1,4,7‐triazacyclononane, 1‐glutaric acid‐5,7 acetic acid (NODAGA) analogue in a preliminary HER2 tumour mouse model. The chelators were conjugated to trastuzumab using the activated esters DOTA mono‐N‐hydroxysuccinimide (NHS) and NODAGA‐NHS. ⁶⁴Cu‐labelling of DOTA‐trastuzumab was studied by varying the amount of DOTA‐trastuzumab used, reaction temperature and time. Full ⁶⁴Cu incorporation could be achieved using a minimum of 10‐µg DOTA‐trastuzumab, but the fastest labelling was obtained after 15 min at room temperature using 25 µg of DOTA‐trastuzumab. In comparison, 80% incorporation was achieved for ⁶⁴Cu‐labelling of NODAGA‐trastuzumab. Both [⁶⁴Cu]DOTA‐trastuzumab and [⁶⁴Cu]NODAGA‐trastuzumab were produced after purification with radiochemical purities of >97%. The tracers were injected into mice with HER2 expressing tumours. The mice were imaged by positron emission tomography and showed high tumour uptake of 3–9% ID/g for both tracers.     

CD44‐Targeted Docetaxel Conjugate for Cancer Cells and Cancer Stem‐Like Cells: A Novel Hyaluronic Acid‐Based Drug Delivery System

A CD44‐targeted macromolecular conjugate of docetaxel was prepared via a pH‐sensitive linkage to hyaluronic acid and was characterized using NMR, gel permeation chromatography, and differential scanning calorimetry. The conjugated species were further evaluated in terms of drug release, cytotoxicity, cellular uptake, cell cycle inhibition, and subacute toxicity in mice. Cellular microscopic studies revealed that CD44‐expressing cells including MCF‐7 cancer stem cells and MDA‐MB‐231 metastatic breast cancer cells had internalized the conjugates via a selective receptor‐mediated mechanism, leading to cell cycle arrest in the G2/M phase. Hyaluronic acid–docetaxel conjugates showed specific toxicity only in CD44‐expressing cells in vitro, along with a decreased risk of neutropenia and dose‐dependent mortality in vivo. Hyaluronic acid–drug conjugates represent a promising and efficient platform for solubilization of sparingly soluble molecules as well as active and selective targeted delivery to cancer cells and cancer stem cells.     

Design and characterization of HER-2-targeted gold nanoparticles for enhanced X-radiation treatment of locally advanced breast cancer

Our purpose was to develop a human epidermal growth factor receptor-2 (HER-2) targeted nanotechnology-based radiosensitizer. HER-2 is overexpressed in 20-30% of all breast cancers and up to 2-fold higher in locally advanced disease (LABC). Trastuzumab was derivatized with a polyethylene glycol (OPSS-PEG-SVA) cross-linker to produce trastuzumab-PEG-OPSS. These immunoconjugates were analyzed by SDS-PAGE, and their immunoreactivity was assessed by flow cytometry using HER-2 overexpressing SK-BR-3 breast cancer cells. Reacting trastuzumab with increasing ratios of PEG resulted in an increase in molecular weight from approximately 148 kDa to 243 kDa, associated with increasing PEG substitution (0.6 to 18.9 PEG chains per trastuzumab). Attachment of approximately 7 PEG chains per trastuzumab resulted in 56% retention in immunoreactivity assessed by flow cytometry. The conjugates were then linked to 30 nm AuNPs. Using a novel (123)iodine-radiotracer based assay that overcomes the current limitations of spectrophotometric quantification of biological molecules on AuNPs we estimate 14.3 ± 2.7 antibodies were attached to each AuNP when 2 × 10(11) AuNPs were reacted with 20 μg of trastuzumab-PEG-OPSS. Specificity of trastuzumab-PEG-AuNPs for HER-2 and internalization in SK-BR-3 cells was demonstrated by comparing the uptake of trastuzumab-PEG-AuNPs or PEG-AuNPs by darkfield microscopy. The ability of trastuzumab-PEG-AuNPs in combination with 300 kVp X-rays to enhance DNA double strand breaks (DSBs) in SK-BR-3 cells was assessed by immunofluorescence using the γ-H2AX assay. γ-H2AX assay results revealed 5.1-fold higher DNA-DSBs with trastuzumab-PEG-AuNPs and X-radiation as compared to treatment with X-radiation alone. The trastuzumab-PEG-AuNPs are a promising targeted nanotechnology-based radiosensitizer for improving LABC therapy. The design and systematic approaches taken to surface modify and characterize trastuzumab-PEG-AuNPs described in this study would have application to other molecularly targeted AuNPs for cancer treatment.     

Design and synthesis of new 2‐anilinoquinolines bearing N‐methylpicolinamide moiety as potential antiproliferative agents

A series of new 2‐anilinoquinolines 6a – o possessing the substantial N‐methylpicolinamide motif at C5 has been designed and synthesized as sorafenib analogs. The antiproliferative activities of the target compounds were preliminarily appraised against a panel of three human cancer cell lines (MCF‐7, SK‐BR3, and HCT116), and a selected array was further tested over a panel of approximately 60 cancer cell lines at NCI at 10 μM concentration. Interestingly, compounds 6c , 6d , 6j , 6k , and 6l showed promising selective anticancer activities (growth inhibition >80%) toward certain cancer cells at 10 μM testing dose. Compounds 6d and 6j were advanced to five‐dose testing mode to determine their GI₅₀ values and compared with our previously reported ureidoquinoline B and sorafenib as reference compounds. The 4‐chloro‐3‐trifluoromethylaniline derivative 6j manifested superior potency than both compound B and sorafenib over eleven and eight cell lines, respectively. It showed GI₅₀ values of 0.36, 0.66, 0.68, and 0.60 μM against the breast MDA‐MB‐468, renal A498, and melanoma SK‐MEL‐5 and UACC‐62 cell lines, respectively. Moreover, both 6d and 6j exerted low cytotoxic effects against HFF‐1 normal cell line. Furthermore, compounds 6d and 6j were tested against both B‐Raf^V600E and C‐Raf kinases and displayed modest inhibitory activities, which were justified by molecular docking study. Compound 6j could serve as a promising candidate for further development of potent anticancer chemotherapeutics.     

Solamargine enhances HER2 expression and increases the susceptibility of human lung cancer H661 and H69 cells to trastuzumab and epirubicin

We have previously demonstrated that solamargine (SM), the major steroidal glycoalkaloid extracted from Chinese herb Solanum plants, reveals down-regulation of HER2 and up-regulation of Fas and tumor necrosis factor receptor (TNFR) expressions, triggers the mitochondria-mediated cell apoptosis pathway, and sensitizes human nonsmall cell lung cancer (NSCLC) H441 and A549 adenocarcinoma cells to chemotherapy. The present study shows that SM enhances HER2 expression in NSCLC large cell carcinoma H661 and small cell lung cancer (SCLC) H69 cells and may increase the susceptibility of the cells to trastuzumab, the humanized anti-HER2 antibody. The combinational treatment of SM and trastuzumab synergistically augments and inhibits H661 and H69 cell proliferation. After treatment with SM, coexpression of HER2 and topoisomerase IIalpha (TOP2A) H661 and H69 cells is more sensitive to the TOP2 inhibitor, epirubicin. The combinatory use of low concentrations of SM with the low-toxic epirubicin accelerated greater apoptotic cell death than each drug did alone in H661 and H69 cells. Relevant studies have shown that HER2 overexpressing cancer cells are more resistant than HER2 low-expressing cells to the chemotherapeutic agent and tumor necrosis factor-induced apoptosis. These investigations have indicated that HER2 overexpression does not suffice to induce intrinsic and pleomorphic drug resistance. The data presented herein suggest that the expression of HER2 did not influence the SM-induced apoptosis of different types of lung cancer cells and that the SM up-regulation of HER2 and TOP2A expressions simultaneously augmented trastuzumab and epirubicin-induced deaths of lung cancer H661 and H69 cells.     

Trastuzumab: in HER2-positive metastatic gastric cancer

Trastuzumab is a recombinant humanized IgG? monoclonal antibody directed against the extracellular domain of the human epidermal growth factor receptor type 2 (HER2) that inhibits HER2-dependent tumour cell proliferation and survival. Proliferation of gastric cancer cells overexpressing HER2 is inhibited by trastuzumab in vitro and in vivo. HER2-positive expression (defined as immunohistochemistry 3+ or fluorescence in situ hybridization-positive) was observed in 22.1% of almost 4000 metastatic gastric cancers in patients who were screened for randomization in the open-label, multicentre, phase III ToGA trial. In patients with HER2-positive metastatic gastric cancer (n?=?584), median overall survival (primary endpoint) was significantly longer for recipients of intravenous trastuzumab plus chemotherapy (comprising cisplatin and either fluorouracil or capecitabine) than in those receiving chemotherapy alone in the ToGA trial. Furthermore, the overall response rate was significantly higher and the median time to disease progression and median time of progression-free survival were also significantly longer with trastuzumab plus chemotherapy than with chemotherapy alone. In general, combination therapy with trastuzumab and chemotherapy was relatively well tolerated in patients with metastatic gastric cancer with no reports of new or unexpected adverse events.     

Role of trastuzumab in adjuvant therapy for locally invasive breast cancer.

PURPOSE: The role of trastuzumab in adjuvant therapy for locally invasive breast cancer is discussed. SUMMARY: Trastuzumab is a humanized monoclonal antibody that binds to the extracellular domain of human epidermal growth factor receptor-2 (HER2). Currently, trastuzumab is indicated for use in HER2-positive patients with metastatic breast cancer. Because trastuzumab specifically targets a receptor that is overexpressed in tumor cells, it is less likely to cause the cytotoxic adverse effects of traditional chemotherapy. Cardiotoxicity has been a major concern, however. Several trials were started to evaluate trastuzumab in the adjuvant setting in patients diagnosed with early-stage breast cancer. The interim results of these trials have shown a promising effect of adjuvant therapy with trastuzumab in improving overall survival, disease-free survival, relapse-free survival, and distant-disease-free survival. CONCLUSION: The use of trastuzumab as adjuvant therapy in patients with HER2-positive breast cancer can lead to increased survival. The appropriateness of trastuzumab therapy should be considered based on HER2 status, cost, and risk of toxicity.     

Aminooxy‐functionalized DOTA for radiolabeling of oxidized antibodies: evaluation of site‐specific 111In‐labeled trastuzumab

Antibody‐targeted nuclear imaging or radiotherapy rely on chemical modification of the polypeptide backbone that may hamper the antigen binding and hence the targeting ability. In this study, we examine a strategy for covalent modification and radiolabeling of monoclonal antibodies utilizing the oligosaccharide moieties on the molecule: the ¹¹¹In‐radiolabeling of the monoclonal antibody trastuzumab (Herceptin®, Roche, Basel, Switzerland), which targets the breast cancer‐related ErbB2/human epidermal growth factor‐2 (HER2) protein. The site‐specific radiolabeled antibody was obtained through mild oxidation of the antibody Fc region with periodate and its conjugation with an aminooxy‐functionalized tetraazacyclododecanetetraacetic acid (DOTA) chelator. It was further evaluated both in vitro with HER2 expressing cells and in vivo with tumor‐bearing mice by single photon emission computed tomography (SPECT), and compared with the DOTA‐N‐hydroxysuccinimide methodology. Such radiolabeled trastuzumab did not show significant difference in stability when compared with lysine‐conjugated antibodies (>95%, 3 days postinjection). They also maintain their immunoreactivity (>90% immunoreactive fraction) and affinity both in vitro (K_d ~ 4–5 nM) and in vivo (12.9 %ID/g in MDA‐MB‐361 tumors). Our results confirm that the formation of a hydrolytically stable oxime linker between a chelating agent and the oxidized N‐glycan residues, away from the variable antigen‐binding sites, is an attractive approach for site‐specific conjugation of antibodies and accessing radiopharmaceuticals and immunotherapeutics. Copyright © 2012 John Wiley & Sons, Ltd.     

Intrathecal trastuzumab (Herceptin) and methotrexate for meningeal carcinomatosis in HER2-overexpressing metastatic breast cancer: a case report

Leptomeningeal carcinomatosis represents a rare manifestation of metastatic breast cancer (MBC). We herewith report on a patient suffering from HER2 overexpressing MBC who received intrathecal methotrexate and trastuzumab for meningeal carcinomatosis. A 48-year-old woman was diagnosed with breast cancer in December 2002. Following surgery, six cycles of adjuvant FE100C plus irradiation and, subsequently for 1 year, trastuzumab were given. As a result of disseminated metastatic spread in October 2005, the patient received whole-brain radiotherapy for symptomatic central nervous system involvement, and was put on several trastuzumab-based combination regimens (capecitabine, vinorelbine, paclitaxel). In June 2006, the patient developed clinical signs of terminal cone involvement with overflow incontinence and paraparesis of the legs. Immediate radiation led to partial relief from clinical symptoms. Subsequently, the patient was put on the tyrosine kinase inhibitor lapatinib and capecitabine (August to October 2007), but on November 6th the patient suffered again from overflow incontinence and weakness of the legs. Failing to respond to lapatinib, the patient received gemcitabine/cisplatin and, additionally, was recommenced on intravenous trastuzumab. Owing to progressive leptomeningeal disease, the patient received repeated doses of intrathecal methotrexate and trastuzumab. Within 2 weeks and four intrathecal treatments, cerebrospinal fluid cytology showed the absence of tumor cells. Moreover, a striking clinical improvement with resolution of the paraparesis of the legs and overflow incontinence was observed. This case report gives details regarding the clinical course of a breast cancer patient who received intrathecal trastuzumab and methotrexate via lumbar puncture for meningeal carcinomatosis of HER2-overexpressing MBC.     

Pharmacologic inhibition of mTOR improves lapatinib sensitivity in HER2-overexpressing breast cancer cells with primary trastuzumab resistance

Lapatinib, a dual EGFR/HER2 kinase inhibitor, is approved for use in patients with trastuzumab-refractory HER2- overexpressing breast cancer. Increased PI3K signaling has been associated with resistance to trastuzumab, although its role in lapatinib resistance remains unclear. The purpose of the current study was to determine if PI3K/mTOR activity affects lapatinib sensitivity. Reduced sensitivity to lapatinib was associated with an inability of lapatinib to inhibit Akt and p70S6K phosphorylation. Transfection of constitutively active Akt reduced lapatinib sensitivity, while kinase-dead Akt increased sensitivity. Knockdown of 4EBP1 also increased lapatinib sensitivity, in contrast to p70S6K knockdown, which did not affect response to lapatinib. Pharmacologic inhibition of mTOR using rapamycin or ridaforolimus increased lapatinib sensitivity and reduced phospho-Akt levels in cells that showed poor response to single-agent lapatinib, including those transfected with hyperactive Akt. Finally, combination mTOR inhibition plus lapatinib resulted in synergistic inhibition of proliferation, reduced anchorage-independent growth, and reduced in vivo tumor growth of HER2- overexpressing breast cancer cells that have primary trastuzumab resistance. Our data suggest that PI3K/mTOR inhibition is critical for achieving optimal response to lapatinib. Collectively, these experiments support evaluation of lapatinib in combination with pharmacologic mTOR inhibition as a potential strategy for inhibiting growth of HER2-overexpressing breast cancers that show resistance to trastuzumab and poor response to lapatinib.     

HER2阳性的进展期胃癌目前和未来的靶向治疗

目前对胃癌中人表皮生长因子受体(2HER2)的预测价值存有争议。当前的治疗指南已经把检测胃癌中HER2状态作为标准化操作。最近,在治疗HER2阳性进展期胃癌中,曲妥珠单抗已经成为一线靶向治疗用药,而原发与继发性药物抵抗则成为主要问题,需要新的治疗策略来克服这种抵抗。许多HER2阳性进展期胃癌患者在接受曲妥珠单抗治疗后都出现疾病进展,必须接受二线方案治疗。新的靶向药物,诸如酪氨酸激酶抑制剂(TKI)拉帕替尼、哺乳动物雷帕霉素靶蛋白(mTOR)通路抑制剂依维莫司、热休克蛋白90(HSP90)抑制剂AUY922、HER二聚化抑制剂帕妥珠单抗以及抗体-药物偶联物曲妥珠单抗-emtansine(T-DM1),在以曲妥珠单抗为基础的一线治疗失败时可以成为二线治疗用药。     

Novel Dithiocarbamic Acid Esters Derived from 6‐Aminomethyl‐4‐anilinoquinazolines and 6‐Aminomethyl‐4‐anilino‐3‐cyanoquinolines as Potent EGFR Inhibitors

Two series of dithiocarbamic acid esters, 4‐anilinoquinazoline‐6‐ylmethylcarbamodithioic acid esters and 3‐cyano‐4‐anilinoquinolin‐6‐ylmethylcarbamodithioic acid esters, were designed and synthesized. The effect of the synthesized compounds on cell proliferation was evaluated by MTT assay against three human cancer cell lines: MDA‐MB‐468, SK‐BR‐3 and HCT‐116. Most of the compounds are equally or more potent than the positive control lapatinib. Three compounds ( 14d , 14h and 14i) were identified as dual inhibitors of the EGFR and ErbB‐2 kinases and two compounds ( 14b and 14c) were identified as multi‐target kinase inhibitors, and they are very worthy of further study. Installation of the dithiocarbamic acid ester group at the 6‐position of 4‐anilinoquinazoline or 3‐cyano‐4‐anilinoquinoline could improve the inhibitory activity. Different dithiocarbamic acid ester groups significantly affect the activities.     

Antibody‐Drug Conjugates: A Clinical Pharmacy Perspective on an Emerging Cancer Therapy

Antibody‐drug conjugates (ADCs) combine highly specific monoclonal antibodies with potent cytotoxic drugs. Their synergy allows for targeted delivery of toxic drugs to cancer cells while sparing systemic exposure. In this review, we focus on the history and clinical applications of ADCs approved by the U.S. Food and Drug Administration (FDA) for the treatment of cancer and highlight new ADCs in the drug development pipeline. Three ADCs have received FDA approval thus far. Gemtuzumab ozogamicin, although withdrawn from the U.S. market, may still be an effective treatment modality in subsets of patients with acute myeloid leukemia. Brentuximab vedotin and ado‐trastuzumab emtansine have shown improved efficacy and safety data compared with standard chemotherapy for the treatment of advanced lymphoma and breast cancer, respectively. With a number of ADCs with promising preliminary data in the clinical trial pipeline, cancer therapy is moving forward from traditional chemotherapy to targeted treatment modalities driven by the specificity of monoclonal antibodies and advancing biotechnology.     

New cancer immunotherapy using autologous lymphocytes activated with trastuzumab

It is well known that trastuzumab (TTZ) is molecular target drug for breast cancer overexpressing human epidermal growth factor receptor 2 (HER2). Novel immunotherapy by human peripheral blood mononuclear cells (PBMCs) activated with TTZ were examined. Proliferation of lymphocytes after adding of TTZ was obtained. Furthermore, lymphocytes activated with TTZ inhibited growth of breast cancer cells in vitro. It is noteworthy that remarkably high cellular cytotoxicity in lymphocytes activated with TTZ compared with that of CD3- and lymphokine (interleukin (IL)2)-activated killer (CD3-LAK) cells commonly used in immunotherapy were revealed.     

Multi‐walled carbon nanotubes induce cytotoxicity and genotoxicity in human lung epithelial cells

The increasing use of nanomaterials in consumer products highlights the importance of understanding their potential toxic effects. We evaluated cytotoxic and genotoxic/oxidative effects induced by commercial multi‐walled carbon nanotubes (MWCNTs) on human lung epithelial (A549) cells treated with 5, 10, 40 and 100 µg ml^?1 for different exposure times. Scanning electron microscopy (SEM) analysis, MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide] and lactate dehydrogenase (LDH) assays were performed to evaluate cytotoxicity. Fpg‐modified comet assay was used to evaluate direct‐oxidative DNA damage. LDH leakage was detected after 2, 4 and 24 h of exposure and viability reduction was revealed after 24 h. SEM analysis, performed after 4 and 24 h exposure, showed cell surface changes such as lower microvilli density, microvilli structure modifications and the presence of holes in plasma membrane. We found an induction of direct DNA damage after each exposure time and at all concentrations, statistically significant at 10 and 40 µg ml^?1 after 2 h, at 5, 10, 100 µg ml^?1 after 4 h and at 10 µg ml^?1 after 24 h exposure. However, oxidative DNA damage was not found. The results showed an induction of early cytotoxic effects such as loss of membrane integrity, surface morphological changes and MWCNT agglomerate entrance at all concentrations. We also demonstrated the ability of MWCNTs to induce early genotoxicity. This study emphasizes the suitability of our approach to evaluating simultaneously the early response of the cell membrane and DNA to different MWCNT concentrations and exposure times in cells of target organ. The findings contribute to elucidation of the mechanism by which MWCNTs cause toxic effects in an in vitro experimental model. Copyright © 2012 John Wiley & Sons, Ltd.