Susan K. Lutgendorf

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 β2 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 β-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.

The influence of bio-behavioural factors on tumour biology: pathways and mechanisms

Epidemiological studies indicate that stress, chronic depression and lack of social support might serve as risk factors for cancer development and progression. Recent cellular and molecular studies have identified biological processes that could potentially mediate such effects. This review integrates clinical, cellular and molecular studies to provide a mechanistic understanding of the interface between biological and behavioural influences in cancer, and identifies novel behavioural or pharmacological interventions that might help improve cancer outcomes.

Adjunctive non-pharmacological analgesia for invasive medical procedures: a randomised trial

BACKGROUND Non-pharmacological behavioural adjuncts have been suggested as efficient safe means in reducing discomfort and adverse effects during medical procedures. We tested this assumption for patients undergoing percutaneous vascular and renal procedures in a prospective, randomised, single-centre study. METHODS 241 patients were randomised to receive intraoperatively standard care (n=79), structured attention (n=80), or self-hypnotic relaxation (n=82). All had access to patient-controlled intravenous analgesia with fentanyl and midazolam. Patients rated their pain and anxiety on 0-10 scales before, every 15 min during and after the procedures. FINDINGS Pain increased linearly with procedure time in the standard group (slope 0.09 in pain score/15 min, p<0.0001), and the attention group (slope 0.04/15 min; p=0.0425), but remained flat in the hypnosis group. Anxiety decreased over time in all three groups with slopes of -0.04 (standard), -0.07 (attention), and -0.11 (hypnosis). Drug use in the standard group (1.9 units) was significantly higher than in the attention and hypnosis groups (0.8 and 0.9 units, respectively). One hypnosis patient became haemodynamically unstable compared with ten attention patients (p=0.0041), and 12 standard patients (p=0.0009). Procedure times were significantly shorter in the hypnosis group (61 min) than in the standard group (78 min, p=0.0016) with procedure duration of the attention group in between (67 min). INTERPRETATION Structured attention and self-hypnotic relaxation proved beneficial during invasive medical procedures. Hypnosis had more pronounced effects on pain and anxiety reduction, and is superior, in that it also improves haemodynamic stability.

Paraneoplastic Thrombocytosis in Ovarian Cancer

BACKGROUND The mechanisms of paraneoplastic thrombocytosis in ovarian cancer and the role that platelets play in abetting cancer growth are unclear. METHODS We analyzed clinical data on 619 patients with epithelial ovarian cancer to test associations between platelet counts and disease outcome. Human samples and mouse models of epithelial ovarian cancer were used to explore the underlying mechanisms of paraneoplastic thrombocytosis. The effects of platelets on tumor growth and angiogenesis were ascertained. RESULTS Thrombocytosis was significantly associated with advanced disease and shortened survival. Plasma levels of thrombopoietin and interleukin-6 were significantly elevated in patients who had thrombocytosis as compared with those who did not. In mouse models, increased hepatic thrombopoietin synthesis in response to tumor-derived interleukin-6 was an underlying mechanism of paraneoplastic thrombocytosis. Tumor-derived interleukin-6 and hepatic thrombopoietin were also linked to thrombocytosis in patients. Silencing thrombopoietin and interleukin-6 abrogated thrombocytosis in tumor-bearing mice. Anti-interleukin-6 antibody treatment significantly reduced platelet counts in tumor-bearing mice and in patients with epithelial ovarian cancer. In addition, neutralizing interleukin-6 significantly enhanced the therapeutic efficacy of paclitaxel in mouse models of epithelial ovarian cancer. The use of an antiplatelet antibody to halve platelet counts in tumor-bearing mice significantly reduced tumor growth and angiogenesis. CONCLUSIONS These findings support the existence of a paracrine circuit wherein increased production of thrombopoietic cytokines in tumor and host tissue leads to paraneoplastic thrombocytosis, which fuels tumor growth. We speculate that countering paraneoplastic thrombocytosis either directly or indirectly by targeting these cytokines may have therapeutic potential. (Funded by the National Cancer Institute and others.).

Stress Hormone–Mediated Invasion of Ovarian Cancer Cells

Purpose: There is growing evidence that stress and other behavioral factors may affect cancer progression and patient survival. The underlying mechanisms for this association are poorly understood. The purpose of this study is to determine the effects of stress-associated hormones norepinephrine, epinephrine, and cortisol on the invasive potential of ovarian cancer cells. Experimental Design: The ovarian cancer cells EG, SKOV3, and 222 were exposed to increasing levels of either norepinephrine, epinephrine, or cortisol, and the in vitro invasive potential was determined using the membrane invasion culture system. Additionally, the effects of these stress hormones on matrix metalloproteinase-2 (MMP-2) and MMP-9 were determined by ELISA. The effects of the β-adrenergic agonist isoproterenol on in vivo tumor growth were determined using nude mice. Results: Stress levels of norepinephrine increased the in vitro invasiveness of ovarian cancer cells by 89% to 198%. Epinephrine also induced significant increases in invasion in all three cell lines ranging from 64% to 76%. Cortisol did not significantly affect invasiveness of the EG and 222 cell lines but increased invasion in the SKOV3 cell line (P = 0.01). We have previously shown that ovarian cancer cells express β-adrenergic receptors. The β-adrenergic antagonist propanolol (1 μmol/L) completely blocked the norepinephrine-induced increase in invasiveness. Norepinephrine also increased tumor cell expression of MMP-2 (P = 0.02 for both SKOV3 and EG cells) and MMP-9 (P = 0.01 and 0.04, respectively), and pharmacologic blockade of MMPs abrogated the effects of norepinephrine on tumor cell invasive potential. Isoproterenol treatment resulted in a significant increase in tumor volume and infiltration in the SKOV3ip1 in vivo model, which was blocked by propranolol. Conclusions: These findings provide direct experimental evidence that stress hormones can enhance the invasive potential of ovarian cancer cells. These effects are most likely mediated by stimulation of MMPs.

Curcumin Inhibits Tumor Growth and Angiogenesis in Ovarian Carcinoma by Targeting the Nuclear Factor-κB Pathway

Purpose: Curcumin, a component of turmeric, has been shown to suppress inflammation and angiogenesis largely by inhibiting the transcription factor nuclear factor-κB (NF-κB). This study evaluates the effects of curcumin on ovarian cancer growth using an orthotopic murine model of ovarian cancer. Experimental Design:In vitro and in vivo experiments of curcumin with and without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8-MDR in athymic mice. NF-κB modulation was ascertained using electrophoretic mobility shift assay. Evaluation of angiogenic cytokines, cellular proliferation (proliferating cell nuclear antigen), angiogenesis (CD31), and apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) was done using immunohistochemical analyses. Results: Curcumin inhibited inducible NF-κB activation and suppressed proliferation in vitro. In vivo dose-finding experiments revealed that 500 mg/kg orally was the optimal dose needed to suppress NF-κB and signal transducers and activators of transcription 3 activation and decrease angiogenic cytokine expression. In the SKOV3ip1 and HeyA8 in vivo models, curcumin alone resulted in 49% (P = 0.08) and 55% (P = 0.01) reductions in mean tumor growth compared with controls, whereas when combined with docetaxel elicited 96% (P < 0.001) and 77% reductions in mean tumor growth compared with controls. In mice with multidrug-resistant HeyA8-MDR tumors, treatment with curcumin alone and combined with docetaxel resulted in significant 47% and 58% reductions in tumor growth, respectively (P = 0.05). In SKOV3ip1 and HeyA8 tumors, curcumin alone and with docetaxel decreased both proliferation (P < 0.001) and microvessel density (P < 0.001) and increased tumor cell apoptosis (P < 0.05). Conclusions: Based on significant efficacy in preclinical models, curcumin-based therapies may be attractive in patients with ovarian carcinoma.

Focal Adhesion Kinase Targeting Using In vivo Short Interfering RNA Delivery in Neutral Liposomes for Ovarian Carcinoma Therapy

Purpose: Focal adhesion kinase (FAK) plays a critical role in ovarian cancer cell survival and in various steps in the metastatic cascade. Based on encouraging in vitro results with FAK silencing, we examined the in vivo therapeutic potential of this approach using short interfering RNA (siRNA) in the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). Experimental Design: Therapy experiments of FAK siRNA with or without docetaxel were done using human ovarian cancer cell lines SKOV3ip1, HeyA8, and HeyA8MDR in nude mice. Additional experiments with a cisplatin-resistant cell line (A2780-CP20) were also done. Assessments of angiogenesis (CD31), cell proliferation (proliferating cell nuclear antigen), and apoptosis (terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling) were done using immunohistochemical analysis. Results: A single dose of FAK siRNA-DOPC was highly effective in reducing in vivo FAK expression for up to 4 days as assayed by Western blot and immunohistochemical analysis. Therapy experiments were started 1 week after injection of the ovarian cancer cells. Treatment with FAK siRNA-DOPC (150 μg/kg twice weekly) reduced mean tumor weight by 44% to 72% in the three cell lines compared with the control group (Ps < 0.05 for HeyA8, A2780-CP20, and SKOV3ip1). When FAK siRNA-DOPC was combined with docetaxel, there was even greater reduction in mean tumor weight in all models (all Ps < 0.05). Similar results were observed in combination with cisplatin. Treatment with FAK siRNA-DOPC plus docetaxel resulted in decreased microvessel density, decreased expression of vascular endothelial growth factor and matrix metalloproteinase-9, and increased apoptosis of tumor-associated endothelial cells and tumor cells. Conclusions: Taken together, these findings suggest that FAK siRNA-DOPC plus docetaxel or platinum might be a novel therapeutic approach against ovarian cancer.

Effect of Interleukin-8 Gene Silencing With Liposome-Encapsulated Small Interfering RNA on Ovarian Cancer Cell Growth

BACKGROUND Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models. METHODS Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided. RESULTS Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P < .001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P < .01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44%; P < .001) and 38% (95% CI = 28% to 48%; P < .001) in the HeyA8 and SKOV3ip1 models, respectively. CONCLUSIONS Increased IL-8 expression is associated with poor clinical outcome in human ovarian carcinoma, and IL-8 gene silencing decreases tumor growth through antiangiogenic mechanisms.

Journaling about stressful events: Effects of cognitive processing and emotional expression

The effects of two journaling interventions, one focusing on emotional expression and the other on both cognitive processing and emotional expression, were compared during 1 month of journaling about a stressful or traumatic event. One hundred twenty-two students were randomly assigned to one of three writing conditions: (a) focusing on emotions related to a trauma or stressor, (b) focusing on cognitions and emotions related to a trauma or stressor, or (c) writing factually about media events. Writers focusing on cognitions and emotions developed greater awareness of the positive benefits of the stressful event than the other two groups. This effect was apparently mediated by greater cognitive processing during writing. Writers focusing on emotions alone reported more severe illness symptoms during the study than those in other conditions. This effect appeared to be mediated by a greater focus on negative emotional expression during writing.

Social Support, Psychological Distress, and Natural Killer Cell Activity in Ovarian Cancer

PURPOSE Psychosocial stress has been related to impaired immunity in cancer patients. However, the extent to which these relationships exist in immune cells in the tumor microenvironment in humans has not been explored. We examined relationships among distress, social support, and natural killer (NK) cell activity in ovarian cancer patients in peripheral-blood mononuclear cells (PBMC), ascitic fluid, and tumor-infiltrating lymphocytes (TIL). PATIENTS AND METHODS Patients awaiting surgery for a pelvic mass suspected of being ovarian cancer completed psychological questionnaires and gave a presurgical sample of peripheral blood. Samples of tumor and ascites were taken during surgery, lymphocytes were then isolated, and NK cytotoxicity and percentage were determined. The final sample, which was confirmed by surgical diagnosis, included 42 patients with epithelial ovarian cancer and 23 patients with benign masses. RESULTS Peripheral NK cell activity was significantly lower among ovarian cancer patients than in patients with benign masses. Among ovarian cancer patients, NK cytotoxicity in TIL was significantly lower than in PBMC or ascitic fluid. Social support was related to higher NK cytotoxicity in PBMC and TIL, adjusting for stage. Distress was related to lower NK cytotoxicity in TIL. A multivariate model indicated independent associations of both distress and social support with NK cell activity in TIL. CONCLUSION Psychosocial factors, such as social support and distress, are associated with changes in the cellular immune response, not only in peripheral blood, but also at the tumor level. These relationships were more robust in TIL. These findings support the presence of stress influences in the tumor microenvironment.