Cellular glycosylation affects Herceptin binding and sensitivity of breast cancer cells to doxorubicin and growth factors

Alterations in protein glycosylation are a key feature of oncogenesis and have been shown to affect cancer cell behaviour perturbing cell adhesion, favouring cell migration and metastasis. This study investigated the effect of N-linked glycosylation on the binding of Herceptin to HER2 protein in breast cancer and on the sensitivity of cancer cells to the chemotherapeutic agent doxorubicin (DXR) and growth factors (EGF and IGF-1). The interaction between Herceptin and recombinant HER2 protein and cancer cell surfaces (on-rate/off-rate) was assessed using a quartz crystal microbalance biosensor revealing an increase in the accessibility of HER2 to Herceptin following deglycosylation of cell membrane proteins (deglycosylated cells Bmax: 6.83 Hz; glycosylated cells Bmax: 7.35 Hz). The sensitivity of cells to DXR and to growth factors was evaluated using an MTT assay. Maintenance of SKBR-3 cells in tunicamycin (an inhibitor of N-linked glycosylation) resulted in an increase in sensitivity to DXR (0.1 µM DXR P<0.001) and a decrease in sensitivity to IGF-1 alone and to IGF-1 supplemented with EGF (P<0.001). This report illustrates the importance of N-linked glycosylation in modulating the response of cancer cells to chemotherapeutic and biological treatments and highlights the potential of glycosylation inhibitors as future combination treatments for breast cancer

Sex hormone supplementation improves breathing and restores respiratory neuroplasticity following C2 hemisection in rats

In addition to loss of sensory and motor function below the level of the lesion, traumatic spinal cord injury (SCI) may reduce circulating steroid hormones that are necessary for maintaining normal physiological function for extended time periods. For men, who comprise nearly 80% of new SCI cases each year, testosterone is the most abundant circulating sex steroid. SCI often results in significantly reduced testosterone production and may result in chronic low testosterone levels. Testosterone plays a role in respiratory function and the expression of respiratory neuroplasticity. When testosterone levels are low, young adult male rats are unable to express phrenic long-term facilitation (pLTF), an inducible form of respiratory neuroplasticity invoked by acute, intermittent hypoxia (AIH). However, testosterone replacement can restore this respiratory neuroplasticity. Complicating the interpretation of this finding is that testosterone may exert its influence in three possible ways: 1) directly through androgen receptor (AR) activation, 2) through conversion to dihydrotestosterone (DHT) by way of the enzyme 5α-reductase, or 3) through conversion to 17β-estradiol (E2) by way of the enzyme aromatase. DHT signals via AR activation similar to testosterone, but with higher affinity, while E2 activates local estrogen receptors. Evidence to date supports the idea that exogenous testosterone supplementation exerts its influence through estrogen receptor signaling under conditions of low circulating testosterone. Here we explored both recovery of breathing function (measured with whole body barometric plethysmography) and the expression of AIH-induced pLTF in male rats following C2-hemisection SCI. One week post injury, rats were supplemented with either E2 or DHT for 7 days. We hypothesized that E2 would enhance ventilation and reveal pLTF following AIH in SCI rats. To our surprise, though E2 did beneficially impact overall breathing recovery following C2-hemisection, both E2 supplementation and DHT restored the expression of AIH-induced pLTF 2 weeks post-SCI

Age and estrogen-associated reductions in hypoxic ventilatory response and chemosensitivity in female rats

Respiratory function is modulated by circulating steroid hormones. In female rats, steroid hormones fluctuate across the normal estrous cycle and decline with age, similar to human menopause. To determine the influence of steroid hormones, and mimic aspects of age-related reductions in hormones, the ovariectomy model is often employed. Ovariectomy (OVX) induces an immediate and persistent decrease in steroid hormones. The current study aimed to interrogate whether the OVX model of hormone reduction impacted specific aspects of respiratory function [chemosensitivity and the hypoxic ventilatory response (HVR)] in a manner consistent with natural age-related declines in hormones. Using barometric plethysmography, three experimental groups of female rats were assessed for HVR, chemosensitivity, and respiratory neural drive during progressive hypoxic challenges (FIO2: 0.15, 0.12, and 0.09): young (3–5 mos. old; in proestrus; n = 10), young OVX (3–5 mos. old; n = 10), and aged (&gt;20 mos. old; n = 10). Our findings indicted that sex hormone loss did not appear to impact chemosensitivity or neural drive. Natural aging, but not OVX, resulted in decreased HVR as well as reduced magnitude in ventilatory output during stepwise hypoxia. Differences in metabolism were important to the interpretation of these results. Collectively, these data support the concept that aging impacts female respiratory function in complex and unique ways that differ from OVX

Trastuzumab-DM1 causes tumour growth inhibition by mitotic catastrophe in trastuzumab-resistant breast cancer cells in vivo

Introduction Trastuzumab is widely used for the treatment of HER2-positive breast cancer. Despite encouraging clinical results, a significant fraction of patients are, or become, refractory to the drug. To overcome this, trastuzumab-DM1 (T-DM1), a newer, more potent drug has been introduced. We tested the efficacy and mechanisms of action of T-DM1 in nine HER2-positive breast cancer cell lines in vitro and in vivo. The nine cell lines studied included UACC-893, MDA-453 and JIMT-1, which are resistant to both trastuzumab and lapatinib. Methods AlamarBlue cell-proliferation assay was used to determine the growth response of breast cancer cell lines to trastuzumab and T-DM1 in vitro. Trastuzumab- and T-DM1-mediated antibody-dependent cellular cytotoxicity (ADCC) was analysed by measuring the lactate dehydrogenase released from the cancer cells as a result of ADCC activity of peripheral blood mononuclear cells. Severe Combined Immunodeficient (SCID) mice were inoculated with trastuzumab-resistant JIMT-1 cells to investigate the tumour inhibitory effect of T-DM1 in vivo. The xenograft samples were investigated using histology and immunohistochemistry. Results T-DM1 was strongly growth inhibitory on all investigated HER2-positive breast cancer cell lines in vitro. T-DM1 also evoked antibody-dependent cellular cytotoxicity (ADCC) similar to that of trastuzumab. Outgrowth of JIMT-1 xenograft tumours in SCID mice was significantly inhibited by T-DM1. Histologically, the cellular response to T-DM1 consisted of apoptosis and mitotic catastrophe, the latter evidenced by an increased number of cells with aberrant mitotic figures and giant multinucleated cells. Conclusions Our results suggest mitotic catastrophe as a previously undescribed mechanism of action of T-DM1. T-DM1 was found effective even on breast cancer cell lines with moderate HER2 expression levels and cross-resistance to trastuzumab and lapatinib (MDA-453 and JIMT-1).BioMed Central Open acces

Trastuzumab emtansine: mechanisms of action and drug resistance

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate that is effective and generally well tolerated when administered as a single agent to treat advanced breast cancer. Efficacy has now been demonstrated in randomized trials as first line, second line, and later than the second line treatment of advanced breast cancer. T-DM1 is currently being evaluated as adjuvant treatment for early breast cancer. It has several mechanisms of action consisting of the anti-tumor effects of trastuzumab and those of DM1, a cytotoxic anti-microtubule agent released within the target cells upon degradation of the human epidermal growth factor receptor-2 (HER2)-T-DM1 complex in lysosomes. The cytotoxic effect of T-DM1 likely varies depending on the intracellular concentration of DM1 accumulated in cancer cells, high intracellular levels resulting in rapid apoptosis, somewhat lower levels in impaired cellular trafficking and mitotic catastrophe, while the lowest levels lead to poor response to T-DM1. Primary resistance of HER2-positive metastatic breast cancer to T-DM1 appears to be relatively infrequent, but most patients treated with T-DM1 develop acquired drug resistance. The mechanisms of resistance are incompletely understood, but mechanisms limiting the binding of trastuzumab to cancer cells may be involved. The cytotoxic effect of T-DM1 may be impaired by inefficient internalization or enhanced recycling of the HER2-T-DM1 complex in cancer cells, or impaired lysosomal degradation of trastuzumab or intracellular trafficking of HER2. The effect of T-DM1 may also be compromised by multidrug resistance proteins that pump DM1 out of cancer cells. In this review we discuss the mechanism of action of T-DM1 and the key clinical results obtained with it, the combinations of T-DM1 with other cytotoxic agents and anti-HER drugs, and the potential resistance mechanisms and the strategies to overcome resistance to T-DM1.BioMed Central open acces