Exercise-induced stress enhances mammary tumor growth in rats: beneficial effect of the hormone melatonin

We hypothesized that intense exercise training (forced swimming for 30 min, 5 days/week) may enhance the progression of mammary carcinogenesis through the involvement of stress hormones, such as catecholamines and prolactin, which can promote breast cancer. After the appearance of the DMBA-induced tumors in Sprague-Dawley rats, the effect was evaluated of exercise-induced stress (with or without administration of the hormone melatonin) on the survival time, tumor multiplicity, and tumor growth until the death of the animals. In a second set of experiments, after one month of exercise, the NK cells count in blood, and the plasma concentrations of catecholamines and prolactin were determined. Although no significant change was found in either the survival time of the rats or the tumor multiplicity, exercise significantly increased the tumor growth rate. Stress was confirmed by the enhanced adrenaline and prolactin concentrations in the blood of the exercised rats. Exercise-induced stress did not change the percentage of NK cells in the tumor-bearing rats. Melatonin counteracted the increased tumor growth, returning the prolactin and adrenaline concentrations to their optimal physiological levels in the exercised tumor-bearing rats, thus confirming an “anti-stress” role of this hormone. In conclusion, intense exercise-induced stress enhances mammary carcinogenesis through the involvement of adrenaline and prolactin. The results also confirmed a role of melatonin as a therapeutic aid against breast cancer in general, and in particular during situations of stress.     

Chronic stress promotes tumor growth and angiogenesis in a mouse model of ovarian carcinoma

Stress can alter immunological, neurochemical and endocrinological functions, but its role in cancer progression is not well understood. Here, we show that chronic behavioral stress results in higher levels of tissue catecholamines, greater tumor burden and more invasive growth of ovarian carcinoma cells in an orthotopic mouse model. These effects are mediated primarily through activation of the tumor cell cyclic AMP (cAMP)-protein kinase A (PKA) signaling pathway by the beta(2) adrenergic receptor (encoded by ADRB2). Tumors in stressed animals showed markedly increased vascularization and enhanced expression of VEGF, MMP2 and MMP9, and we found that angiogenic processes mediated the effects of stress on tumor growth in vivo. These data identify beta-adrenergic activation of the cAMP-PKA signaling pathway as a major mechanism by which behavioral stress can enhance tumor angiogenesis in vivo and thereby promote malignant cell growth. These data also suggest that blocking ADRB-mediated angiogenesis could have therapeutic implications for the management of ovarian cancer.     

Overexpression of VEGF189 in breast cancer cells induces apoptosis via NRP1 under stress conditions

The existence of multiple VEGF-A isoforms raised the possibility that they may have distinct functions in tumor growth. We have previously published that VEGF189 and VEGF165 contribute to breast cancer progression and angiogenesis, but VEGF165 induced the most rapid tumor uptake. Since VEGF165 has been described as a survival factor for breast tumor cells, we questioned here the effects of VEGF189 on the survival/apoptosis of MDA-MB-231 cells. We used clones which overexpress VEGF189 (V189) or VEGF165 (V165) isoforms and compared them to a control one (cV). Overexpression of VEGF189 resulted in increased cell apoptosis, as determined by Annexin-V apoptosis assay, under serum starvation and doxorubicin treatment, while VEGF 165 was confirmed to be a survival factor. Since MDA-MB-231 highly express NRP1 (a co-receptor for VEGF-A), we used short hairpin RNA (shRNA) to knockdown NRP1 expression. V189shNRP1 clones were characterized by reduced apoptosis and higher necrosis, as compared to V189shCtl, under stress conditions. Unexpectedly, NRP1 knock-down had no effect on the survival or apoptosis of V165 cells. VEGF189 showed greater affinity towards NRP1 than VEGF165 using a BIAcore binding assay. Finally, since endogenously produced urokinase-type plasminogen (uPA) has been found to prevent apoptosis in breast cancers, we analyzed the level of uPA activity in our clones. An inhibition of uPA activity was observed in V189shNRP1 clones. Altogether, these results suggest a major role of NRP1 in apoptosis induced by VEGF189 in stress conditions and confirm VEGF165 as a survival factor.     

Cardio-oncology: an overview on outpatient management and future developments

Recent advances in the early detection and treatment of cancer have led to increasing numbers of cancer survivors worldwide. Nonetheless, despite major improvements in the outcome of these patients, long-term side effects of radio- and chemotherapy affect both patient survival and quality of life, independent of the oncological prognosis. Chemotherapy-related cardiac dysfunction is one of the most notorious short-term side effects of anticancer treatment, occurring in ~10% of patients. Progression to overt heart failure carries a strikingly poor prognosis with a 2-year mortality rate of 60%. Early detection of left ventricular damage by periodic monitoring and prompt initiation of heart failure treatment is key in improving cardiovascular prognosis. To meet the growing demand for a specialised interdisciplinary approach for the prevention and management of cardiovascular complications induced by cancer treatment, a new discipline termed cardio-oncology has evolved. However, an uniform, multidisciplinary approach is currently lacking in the Netherlands. This overview provides an introduction and comprehensive summary of this emerging discipline and offers a practical strategy for the outpatient management of this specific patient population.     

Rich Medium Composition Affects Escherichia coli Survival,Glycation, and Mutation Frequency during Long-Term Batch Culture

Bacteria such as Escherichia coli are frequently grown to high density to produce biomolecules for study in the laboratory. To achieve this, cells can be incubated in extremely rich media that increase overall cell yield. In these various media, bacteria may have different metabolic profiles, leading to changes in the amounts of toxic metabolites produced. We have previously shown that stresses experienced during short-term growth can affect the survival of cells during the long-term stationary phase (LTSP). Here, we incubated cells in LB, 2× yeast extract-tryptone (YT), Terrific Broth, or Super Broth medium and monitored survival during the LTSP, as well as other reporters of genetic and physiological change. We observe differential cell yield and survival in all media studied. We propose that differences in long-term survival are the result of changes in the metabolism of components of the media that may lead to increased levels of protein and/or DNA damage. We also show that culture pH and levels of protein glycation, a covalent modification that causes protein damage, affect long-term survival. Further, we measured mutation frequency after overnight incubation and observed a correlation between high mutation frequencies at the end of the log phase and loss of viability after 4 days of LTSP incubation, indicating that mutation frequency is potentially predictive of long-term survival. Since glycation and mutation can be caused by oxidative stress, we measured expression of the oxyR oxidative stress regulator during log-phase growth and found that higher levels of oxyR expression during the log phase are consistent with high mutation frequency and lower cell density during the LTSP. Since these complex rich media are often used when producing large quantities of biomolecules in the laboratory, the observed increase in damage resulting in glycation or mutation may lead to production of a heterogeneous population of plasmids or proteins, which could affect the quality of the end products yielded in some laboratory experiments.     

Engineered Salmonella typhimurium expressing E7 fusion protein, derived from human papillomavirus, inhibits tumor growth in cervical tumor-bearing mice

The tumor-suppressing effects of SipB160/HPV16 E7 fusion protein, derived from human papillomavirus, and expressed in Salmonella enterica serovar typhimurium, were evaluated in a cervical cancer model. The expressed E7 protein resulted in efficacious cytotoxicity and tumor growth retardation in TC-1 cervical cancer cells. In addition, in mice bearing TC-1 tumors, live cells of Salmonella expressing HPV16 E7 were administered orally and induced immune responses through interferon-gamma and tumor necrosis factor-alpha cytokine secretion and also suppressed tumor growth (45 %) and prolonged survival (70 %) compared with the control group. These results suggested that the SipB160/HPV16 E7 fusion protein may be a candidate cancer therapeutic agent.     

Intratumoral treatment of smaller mouse neuroblastoma tumors with a recombinant protein consisting of IL-2 linked to the Hu14.18 antibody increases intratumoral CD8+ T and NK cells and improves survival

Hu14.18-IL2 is an immunocytokine (IC) consisting of human IL-2 linked to hu14.18 mAb, which recognizes GD2 disialoganglioside. Phase II clinical trials of intravenous-hu14.18-IL2 (IV-IC) in neuroblastoma and melanoma are underway, and have already demonstrated activity in neuroblastoma. In our Phase II trial, lower neuroblastoma burden at the time of treatment was associated with a greater likelihood of clinical response to IV-IC. We have previously shown that intratumoral-hu14.18-IL2 (IT-IC) compared to IV-IC results in enhanced local and systemic antitumor activity in tumor-bearing mice. We utilized a mouse model to investigate the impact of tumor burden on hu14.18-IL2 treatment efficacy in IV- versus IT-treated animals. Studies presented here describe the analyses of tumor burden at the initiation of treatment and its effects on treatment efficacy, survival, and tumor-infiltrating leukocytes in A/J mice bearing subcutaneous NXS2 neuroblastoma. We show that smaller tumor burden at treatment initiation is associated with increased infiltration of NK and CD8+ T cells and increased overall survival. NXS2 tumor shrinkage shortly after completion of the 3 days of hu14.18-IL2 treatment is necessary for long-term survival. This model demonstrates that tumor size is a strong predictor of hu14.18-IL2-induced lymphocyte infiltration and treatment outcome.     

Antibiotic bedaquiline effectively targets growth,survival and tumor angiogenesis of lung cancer through suppressing energy metabolism

Tumor angiogenesis plays essential roles during lung cancer progression and metastasis. Therapeutic agent that targets both tumor cell and vascular endothelial cell may achieve additional anti-tumor efficacy. We demonstrate that bedaquiline, a FDA-approved antibiotic drug, effectively targets lung cancer cells and angiogenesis. Bedaquiline dose-dependently inhibits proliferation and induces apoptosis of a panel of lung cancer cell lines regardless of subtypes and molecular heterogeneity. Bedaquiline also inhibits capillary network formation of human lung tumor associated-endothelial cell (HLT-EC) on Matrigel and its multiple functions, such as spreading, proliferation and apoptosis, even in the presence of vascular endothelial growth factor (VEGF). We further demonstrate that bedaquiline acts on lung cancer cells and HLT-EC via inhibiting mitochondrial respiration and glycolysis, leading to ATP reduction and oxidative stress. Consistently, oxidative damage on DNA, protein and lipid were detected in cells exposed to bedaquiline. Importantly, the results obtained in in vitro cell culture are reproducible in in vivo xenograft lung cancer mouse model, confirming that bedaquiline suppresses lug tumor growth and angiogenesis, and increases oxidative stress. Our findings demonstrating that energy depletion is effectively against lung tumor cells and angiogenesis. Our work also provide pre-clinical evidence to repurpose antibiotic bedaquiline for lung cancer treatment.     

Long-term survival after adoptive bone marrow T cell therapy of advanced metastasized breast cancer: follow-up analysis of a clinical pilot trial

Background

The bone marrow (BM) of breast cancer patients harbors tumor-reactive memory T cells (TCs) with therapeutic potential. We recently described the immunologic effects of adoptive transfer of ex vivo restimulated tumor-reactive memory TCs from the BM of 12 metastasized breast cancer patients in a clinical phase-I study. In this trial, adoptive T cell transfer resulted in the occurrence of circulating tumor antigen-reactive type-1 TCs. We here describe the long-term clinical outcome and its correlation with tumor-specific cellular immune response in 16 metastasized breast cancer patients, including 12 included in the original study.

Methods

Sixteen metastatic breast cancer patients with preexisting tumor-reactive BM memory TCs were included into the study. The study protocol involved one transfusion of TCs which were reactivated in vitro with autologous dendritic cells pulsed with lysates of MCF-7 breast cancer cells as source of tumor antigens. The presence of tumor-reactive memory TCs was analyzed by IFN-γ ELISpot assays.

Results

Tumor-reactive memory TCs in the peripheral blood were induced de novo in 7/16 patients (44 %) after adoptive TC transfer. These patients were considered immunologic responders to the therapy. Positive adoptive immunotherapy (ADI) response was observed significantly more often in patients without bone metastases (p = 0.0051), in patients with high levels of tumor-reactive BM TCs prior to therapy (p = 0.036) and correlated significantly with the estimated numbers of transferred tumor-reactive TCs (p = 0.0021). After the treatment, we observed an overall median survival of 33.8 months in the total cohort with three patients alive at last follow-up and more than 7 years after ADI. Numbers of transferred tumor-reactive TCs correlated significantly with the overall survival of patients (p = 0.017). Patients with an immunologic response to ADI in the peripheral blood had a significantly longer median survival than nonresponders (median survival 58.6 vs. 13.6 months; p = 0.009).

Conclusion

In metastasized breast cancer patients, adoptive transfer of BM TCs can induce the presence of tumor antigen-reactive type-1 TCs in the peripheral blood. Patients with immunologic response after ADI show a significantly longer overall survival. Patients with bone metastases significantly less frequently respond to the treatment and, therefore, might not be optimal candidates for ADI. Although the present study does not yet prove the therapeutic effect of ADI, these findings shed light on the relation between immune response and cancer prognosis and suggest that transfer of reactivated BM TCs might bear therapeutic potential.     

Apatinib,a novel tyrosine kinase inhibitor,suppresses tumor growth in cervical cancer and synergizes with Paclitaxel

Apatinib is a novel tyrosine kinase inhibitor that targets VEGFR2 signal and exhibits potent anti-tumor effects in human cancers. In this study, we aim to investigate the efficacy of Apatinib in cervical cancer. The protein expression of VEGFR2 and its relationships with clinical parameters were investigated in a panel of cervical cancer patients. In vitro, a series of experiments were performed to detect the effects of Apatinib on the proliferation, apoptosis and cell cycle in cervical cancer cells. Both the immortalized cell lines and primary cultured tissues were used to investigate the synergy between Apatinib and chemotherapeutic drugs. The in vivo effects of Apatinib were validated in a nude mouse model. Compared to that in normal cervix, VEGFR2 protein was significantly upregulated in cervical cancer tissues (P < 0.001); this was positively correlated with advanced tumor stage, lymph node metastasis, and a poor prognosis. In vitro, Apatinib markedly induced apoptosis and G1-phase arrest, suppressed cell growth, and decreased colony formation ability. We also found that primary cancer tissues with higher level of VEGFR2 were much more sensitive to Apatinib. Further, we proved that Apatinib significantly increased the sensitivity to Paclitaxel in cervical cancer cells and the mouse model. Collectively, we firstly report the anti-tumor efficacy of Apatinib in cervical cancer. Moreover, Apatinib synergized with Paclitaxel to achieve more significant suppression on tumor growth, proposing that Apatinib might be a potent drug for cervical cancer.     

Autophagy,citrullination and cancer

A cell needs to maintain a balance between biosynthesis and degradation of cellular components to maintain homeostasis. There are 2 pathways, the proteasome, which degrades short-lived proteins, and the autophagy/lysosomal pathway, which degrades long-lived proteins and organelles. Both of these pathways are also involved in antigen presentation or the effective delivery of peptides to MHC molecules for presentation to T cells. Autophagy (macroautophagy) is a key player in providing substantial sources of citrullinated peptides for loading onto MHC-II molecules to stimulate CD4⁺ T cell responses. Stressful conditions in the tumor microenvironment induce autophagy in cancer cells as a mechanism to promote their survival. We therefore investigated if citrullinated peptides could stimulate CD4⁺ T cell responses that would recognize these modifications produced during autophagy within tumor cells. Focusing on the intermediate filament protein VIM (vimentin), we generated citrullinated VIM peptides for immunization experiments in mice. Immunization with these peptides induced CD4⁺ T cells in response to autophagic tumor targets. Remarkably, a single immunization with modified peptide, up to 14 d after tumor implant, resulted in long-term survival in 60% to 90% of animals with no associated toxicity. These results show how CD4⁺ cells can mediate potent antitumor responses against modified self-epitopes presented on tumor cells, and they illustrate for the first time how the citrullinated peptides produced during autophagy may offer especially attractive vaccine targets for cancer therapy.     

Age and rest–activity rhythm as predictors of survival in patients with newly diagnosed lung cancer

Disruption of the rest–activity rhythm in patients with lung cancer can accelerate cancer progression and affect survival. Rest–activity rhythm changes with age. Therefore, we investigated the effects of rest–activity rhythm and age on patients’ survival. A total of 84 patients with lung cancer were recruited, then separated into two groups; younger patients aged under 65 years or elderly patients aged 65 and over. The dichotomy index (I < O) was used to estimate the rest–activity rhythm measured through the actigraphy motion detector. Cox proportional hazards models were adopted to investigate the effects of different variables on the patients’ survival. After adjusting for confounding, the risk of earlier mortality in the younger patients with disrupted I < O were 2.52 (95%CI = 1.09–5.82) times higher than that in the younger patients with robust I < O (p = 0.03), the risk of earlier mortality in the elderly patients with disrupted I < O was 4.08 (95%CI = 1.91–8.68) times higher than that in the elderly patients with robust I < O (p < 0.001). Therefore, age and I < O influence the survival period of patients with lung cancer. Moreover, disrupted I < O has a substantial influence on elderly patients. In conclusion, aging and disrupted rest–activity rhythm negatively and jointly influenced the survival period of the patients with lung cancer and significantly increased their death risk.     

DNA methylation of apoptosis genes in rectal cancer predicts patient survival and tumor recurrence

Deregulation of the apoptotic pathway, one of the hallmarks of tumor growth and -progression, has been shown to have prognostic value for tumor recurrence in rectal cancer. In order to develop clinically relevant biomarkers, we studied the methylation status of promoter regions of key apoptosis genes in rectal cancer patients, using methylation-sensitive restriction enzymes. DNA was extracted from fresh-frozen tumor tissues of 49 stage I-III rectal cancer patients and 10 normal rectal tissues. The results of this pilot study were validated in 88 stage III tumor tissues and 18 normal rectal tissues. We found that methylation of the intrinsic apoptotic pathway genes Apaf1, Bcl2 and p53 correlated with the apoptotic status (M30) of the tumor. Combined survival analyses of these three genes, based on the number of genes showing high methylation (all low, 1 high, 2 high or all high), showed shorter patient survival and recurrence-free periods with an increasing number of methylated markers. Multivariate analyses showed significant differences for overall survival (p = 0.01; HR = 0.28 (0.09–0.83)), cancer-specific survival (p = 0.004; HR = 0.13 (0.03–0.67)) and distant recurrence-free survival (p = 0.001; HR = 0.22(0.05–0.94)). The shortest survival was observed for patients showing low methylation of all markers, which—as was expected—correlated with high apoptosis (M30), but also with high proliferation (Ki-67). The study of epigenetic regulation of apoptosis genes provides more insight in the tumorigenic process in rectal cancer and might be helpful in further refining treatment regimens for individual patients.     

The clinical effect study on malignant tumors with chronoradiotherapy

Like normal cells, tumor cells are also rhythmic. Chronochemotherapy has been proven to be effective in clinic, yet there are still some controversies about the efficacy and radiotherapy-related toxicity of chronoradiotherapy. This study was designed to investigate the therapy-related toxicity and short-term effect of four types of malignant tumor patients who were treated with radiotherapy at two different times. 121 cases of malignant tumor patients with radiotherapy indications were randomly divided into morning radiotherapy (MR) group and evening radiotherapy (ER) group, who received an identical procedure of radiotherapy at 9?am or 9 pm, respectively. The effects of radiotherapy were evaluated by short-term effect and therapy-related toxicity in this study. The efficacy rates of radiotherapy of lung cancer and nasopharyngeal carcinoma in ER groups were higher than those in MR groups, but the difference was not statistically significant (P > 0.05); the efficacy rates of radiotherapy of cervical and esophageal cancers in ER groups were significantly higher than those in MR groups (P < 0.05). The incidences of radiotherapy-related toxicity in these 4 types of malignant tumor patients were significantly lower in ER groups (P < 0.05), compared with MR groups. It suggests that irradiation in the evening could increase the efficacy in cervical and esophageal cancer patients and reduce the incidence of radiotherapy-related toxicity in these four malignant tumor patients.     

Synergistic Effect of CTLA-4 Blockade and Cancer Chemotherapy in the Induction of Anti-Tumor Immunity

Several chemotherapeutics exert immunomodulatory effects. One of these is the nucleoside analogue gemcitabine, which is widely used in patients with lung cancer, ovarian cancer, breast cancer, mesothelioma and several other types of cancer, but with limited efficacy. We hypothesized that the immunopotentiating effects of this drug are partly restrained by the inhibitory T cell molecule CTLA-4 and thus could be augmented by combining it with a blocking antibody against CTLA-4, which on its own has recently shown beneficial clinical effects in the treatment of patients with metastatic melanoma. Here we show, using two non-immunogenic murine tumor models, that treatment with gemcitabine chemotherapy in combination with CTLA-4 blockade results in the induction of a potent anti-tumor immune response. Depletion experiments demonstrated that both CD4⁺ and CD8⁺ T cells are required for optimal therapeutic effect. Mice treated with the combination exhibited tumor regression and long-term protective immunity. In addition, we show that the efficacy of the combination is moderated by the timing of administration of the two agents. Our results show that immune checkpoint blockade and cytotoxic chemotherapy can have a synergistic effect in the treatment of cancer. These results provide a basis to pursue combination therapies with anti-CTLA-4 and immunopotentiating chemotherapy and have important implications for future studies in cancer patients. Since both drugs are approved for use in patients our data can be immediately translated into clinical trials.     

Metabolic control by dehydroascorbic acid: Questions and controversies in cancer cells

For a long time, the effect of vitamin C on cancer cells has been a controversial concept. From Linus Pauling's studies in 1976, it was proposed that ascorbic acid (AA) could selectively kill tumor cells. However, further research suggested that vitamin C has no effect on tumor survival. In the last decade, new and emerging functions for vitamin C have been discovered using the reduced form, AA, and the oxidized form, dehydroascorbic acid (DHA), independently. In this review, we summarized the latest findings related to the effects of DHA on the survival and metabolism of tumor cells. At the same time, we put special emphasis on the bystander effect and the recycling capacity of vitamin C in various cellular models, and how these concepts can affect the experimentation with vitamin C and its therapeutic application in the treatment against cancer.     

A cancer chemopreventive agent silibinin, targets mitogenic and survival signaling in prostate cancer

There are many epigenetic variables that affect the biological responses of autocrine, paracrine and endocrine regulatory molecules, which determine the growth and development of different cancers including prostate cancer (PCA). One of the focuses of the current cancer chemoprevention studies is the search for non-toxic chemopreventive agents that inhibit mitogenic and cell survival signaling in cancer cells. In general, advanced stage cancer cells harbor many constitutively active mitogenic signaling and anti-apoptotic mechanisms, which make them less dependent on external growth factors as well as resistant to chemotherapeutic agents. In this regard, silibinin (a naturally occurring flavanone) has shown the pleiotropic anticancer effects in different cancer cells. Our extensive studies with PCA have shown that inhibition of mitogenic and cell survival signaling, such as epidermal growth factor receptor, insulin-like growth factor receptor type I and nuclear factor kappa B signaling are the most likely molecular targets of silibinin's efficacy in PCA. We have observed that silibinin inhibits prostate tumor growth in animal models without any apparent signs of toxicity. At the same time, silibinin is also physiologically available in different organs of the body including plasma and prostate, which is generally required for the pharmacological dosing and translational mechanistic studies of the compound. There are substantial amount of data to support the inhibitory effect of silibinin on mitogenic and cell survival signaling in PCA, which are reviewed in the present communication.