Int J Clin Oncol DOI 10.1007/s10147-014-0779-y
ORIGINAL ARTICLE
Fibrates protect against vascular endothelial dysfunction induced by paclitaxel and carboplatin chemotherapy for cancer patients: a pilot study Ayako Watanabe · Akiko Tanabe · Risa Maruoka · Kiyoko Nakamura · Koji Hatta · Yoshihiro J. Ono · Yoshito Terai · Masahide Ohmichi
Received: 29 May 2014 / Accepted: 10 December 2014 © Japan Society of Clinical Oncology 2014
Abstract Background Although we previously demonstrated that paclitaxel and carboplatin chemotherapy (TCchem) is associated with vascular toxicities, the underlying mechanisms remain unclear. Cisplatin is known to inhibit PPARα following microvascular damage to the kidneys. The primary aim of this study was to evaluate whether TCchem induces vascular endothelial dysfunction via systemic PPARα deficiency. In addition, human umbilical vein endothelial cells (HUVECs) were used to elucidate the mechanisms responsible for TCchem-induced vascular toxicities. Methods This study enrolled 45 gynecological cancer patients with normal lipid profiles who underwent surgical treatment followed by TCchem. The elevated triglyceride (TG) group included patients (n = 19) who exhibited hypertriglyceridemia during TCchem, and the stable TG group (n = 15) included patients with a normal TG level. Eleven patients exhibiting hypertriglyceridemia during TCchem were administered bezafibrate (fibrate group). Endothelial dysfunction was evaluated based on flowmediated dilation (FMD) values and serum pentraxin-3 levels measured before TCchem and immediately after the final TCchem. HUVECs were used to elucidate the biological mechanisms underlying the endothelial dysfunction induced by TCchem.
Electronic supplementary material The online version of this article (doi:10.1007/s10147-014-0779-y) contains supplementary material, which is available to authorized users. A. Watanabe · A. Tanabe (*) · R. Maruoka · K. Nakamura · K. Hatta · Y. J. Ono · Y. Terai · M. Ohmichi Department of Obstetrics and Gynecology, Osaka Medical College, 2‑7 Daigaku‑machi, Takatsuki, Osaka 569‑8686, Japan e-mail: [email protected]‑med.ac.jp
Results The administration of TCchem induced hypertriglyceridemia in 66 percent of the participants, and bezafibrate reduced the serum TG levels. Meanwhile, the decrease in flow-mediated dilatation (%FMD) induced by TCchem improved following treatment with bezafibrate. The serum pentraxin-3 level increased rapidly after TCchem and decreased following bezafibrate treatment. An in vitro examination demonstrated TCchem attenuated nitric oxide production and PPARα activity in HUVECs, which was partially improved by treatment with bezafibrate. Conclusion Bezafibrate prevents endothelial dysfunction induced by TCchem via TG-dependent and TG-independent mechanisms. Keywords Bezafibrate · Platinum-based chemotherapy · Carboplatin · Paclitaxel · Gynecological cancer · Triglyceride · FMD Abbreviations PtChem Platinum-based chemotherapy TG Triglycerides TCchem Paclitaxel and carboplatin combination chemotherapy FMD Flow-mediated dilation PPARα Peroxisome proliferator-activated receptor-α OC Ovarian cancer EC Endometrial cancer CVD Cardiovascular disease LDL Low-density lipoprotein HDL High-density lipoprotein hsCRP High-sensitivity C-reactive protein CDDP Cisplatin CBDCA Carboplatin PTX Paclitaxel
13
Fig. 1 Consort diagram explaining the recruitment process for each group. OC ovarian cancer, EC endometrial cancer, TG triglycerides, TC paclitaxel and carboplatin chemotherapy
Int J Clin Oncol
Screening Period (n=28) (OC n=14, EC n=14)
Intervenon Period (n=17) (OC n=9, EC n=8)
TC chemotherapy
Hypertriglyceridemia detected during TC Administraon of bezafibrate
Hypertriglyceridemia (OC n=10, EC n=9)
Elevated TG group (n=19) OC n=10 EC n=9
Introduction Cancer survivors are living longer after their initial diagnosis due to earlier diagnoses and improvements in treatment. Epithelial ovarian cancer (OC) and uterine endometrial cancer (EC) are major gynecological malignancies. Patients with these diseases generally undergo surgery, including bilateral oophorectomy, followed by the administration of platinum-based chemotherapy (PtChem). Although the acute adverse effects of combination chemotherapy have been well defined, recent research has focused on the late effects of PtChem. The long-term toxicity of chemotherapy is important due to the prolonged expected lifespan of these patients. Previous reports have suggested that PtChem is associated with vascular toxicities and serious vascular complications (e.g., myocardial infarction, stroke, and thromboembolic disease) [1–4] in testicular cancer survivors. Although the precise mechanisms remain unclear, several reports have demonstrated that cancer survivors who received chemotherapy showed increased plasma levels of endothelial and inflammatory markers, which might progress to atherosclerosis [5, 6], increased risks of hypertension [7–9], and dyslipidemia [7, 9]. We previously demonstrated that TCchem induces a significant increase in arterial stiffness 12 months after treatment and that the %FMD rapidly decreases after TCchem infusion [10]. Interestingly, our previous data demonstrated that serum triglyceride levels are significantly elevated in patients treated with TCchem. Portilla and colleagues reported a significant reduction in proximal tubule fatty acid oxidation leading to the accumulation of toxic fatty acid amphiphiles in patients with PtChem-induced acute renal failure [11–13]. A recent
13
Stable TG (OC n=4, EC n=5)
Stable TG (OC n=4, EC n=2)
Stable TG group (n=15) OC n=8 EC n=7
Fibrate group (n=11) OC n=5 EC n=6
study showed a threefold increase in the serum triglyceride levels on day 3 in mice treated with cisplatin, in addition to the accumulation of triglycerides in the urine and kidney tissue [14]. These metabolic changes and the degree of microvascular damage in the kidneys were improved by the administration of peroxisome proliferator-activated receptor-α (PPARα) ligands, including bezafibrate. These previous studies established that cisplatin deactivates PPARα in cisplatin-induced acute renal failure. There are currently no reports that have elucidated whether TCchem induces the development of systemic PPARα deficiency. Hence, the primary aim of the current pilot study was to evaluate whether TCchem induces vascular endothelial dysfunction via systemic PPARα deficiency. Furthermore, HUVECs were used to elucidate the mechanisms responsible for these effects based on in vitro examinations.
Methods Patients All patients evaluated in this study were under treatment at the Department of Obstetrics and Gynecology of Osaka Medical College Hospital. This study was a prospective study approved by the Institutional Review Board of Osaka Medical College. Written informed consent was obtained from all participants. The study population included 45 gynecological cancer patients who received adjuvant TCchem after surgical treatment according to established protocols between December 2006 and November 2012 (Fig. 1). We compared the following two periods: December 2006 to December 2009 as the screening
Int J Clin Oncol 0
Stable TG group (n=15) OC n=8 EC n=7 Elevated TG group (n=19) OC n=10 EC n=9
1
6
TC
Surgery
Fig. 2 Experimental design. OC ovarian cancer; EC endometrial cancer; TG triglycerides; TC paclitaxel and carboplatin combination chemotherapy; FMD flow-mediated dilation; after chemo: immediately after the final course of chemotherapy; A fasting blood sample collection
TC
TC
Fibrate group (n=11) OC n=5 EC n=6
Bezafibrate A FMD Blood Samples at baseline
period, and January 2010 to November 2012 as the intervention period. In the screening period, the patients in the surgery + TC group (n = 28) evaluated in our previous prospective study [10] were divided into the following two groups: the elevated TG group, including patients who exhibited hypertriglyceridemia (>150 mg/dl) under TCchem; and the stable TG group, including patients who exhibited a normal serum level of triglycerides (≤150 mg/ dl) under TCchem. In the intervention period, 17 patients were newly included. Blood samples were collected to monitor myelosuppression and fasting lipid profiles in the morning of each course of TCchem (indicated as ‘A’ in Fig. 2). In patients who exhibited hypertriglyceridemia (>150 mg/dl) twice during the study period, treatment with bezafibrate® (400 mg/day) was started and continued until the end of the planned adjuvant treatment period. Bezafibrate acts as a synthetic ligand for PPARα, which is mainly expressed in the liver, skeletal muscle, heart, and vascular endothelial cells [15]. The activation of PPARα results in plasma clearance of atherogenic triglyceride-rich lipoproteins [16, 17]. Participants who had received prior treatment for cancer and those with a history of cardiovascular disease or decompensated diabetes were excluded from this study. The exclusion criteria also included a history of clinically significant gastrointestinal, liver, or gallbladder disease, treatment with lipid- or glucose-lowering agents, and the use of hormone replacement therapy within the previous 1 year.
TC
A
A
A FMD Blood Samples at a er chemo
Treatment design A complete medical history was taken and a physical exam was performed in all patients. Pulmonary function tests and an electrocardiogram (ECG) were also performed, and the participants with abnormalities were excluded from this study. The patients underwent total hysterectomy, bilateral salpingo-oophorectomy, pelvic/para-aortic lymphadenectomy, and omentectomy, including the complete removal of all visible tumor tissue. TCchem was administered in all patients according to established protocols, after performing tumor staging using the International Federation of Gynecology and Obstetrics (FIGO) nomenclature. Briefly, patients were premedicated with dexamethasone (20 mg intravenously) for 30 min before the start of paclitaxel infusion. Both a histamine receptor type I blocker, diphenhydramine (50 mg orally), and a type II blocker, ranitidine (50 mg intravenously), were administered 30 min before paclitaxel infusion. Next, the patients received paclitaxel (175 mg/m2) intravenously for 3 h with monitoring of their vital signs (heart rate, blood pressure, pulse, and respiratory rate), followed by carboplatin (AUC 5 mg/ml) intravenously for 1 h on day 1 every 3 weeks for a total of six courses. The carboplatin dose was calculated using the Calvert formula: carboplatin dose (in mg) = AUC × (GFR + 25), where GFR is the glomerular filtration rate, estimated using the Jelliffe formula. Toxicity was assessed at each treatment cycle according to the National Cancer Institute Common Terminology
13
Criteria (version 3.0). Grade 3/4 hematologic toxicities associated with TCchem were managed with a dose reduction of carboplatin to an AUC of 4 or delay in administration for 7 days. Grade 2 or higher signs of peripheral neuropathy were managed with a dose reduction of paclitaxel to 75 %. If the above mentioned toxicities recurred after the first dose reduction or delay in the schedule, the carboplatin and paclitaxel dosages were again reduced to AUC = 3 and 75 %, respectively, or the schedule for chemotherapy was delayed. None of the patients in the current study required dose reduction, although 91.1 % of the patients were treated with a delay in the dosing schedule. All patients received a total of six courses of treatment. Study design The study design is shown in Fig. 2. %FMD and serum levels of pentraxin-3 and high-sensitivity C-reactive protein (hsCRP) were evaluated just before the first course of TCchem (baseline) and immediately after the final course of TCchem (after chemo). Details of the procedure used to measure the FMD values are described in the next paragraph. The levels of pentraxin-3 and hsCRP were measured using an enzyme-linked immunosorbent assay (ELISA) at the central clinical laboratory of Osaka Medical College. Measurement of the %FMD of the brachial artery In order to assess brachial %FMD, the right brachial artery diameter was measured on two-dimensional ultrasound images using a 7.0-MHz linear artery transducer and a ProSound SSD-Alpha 10 system (Hitachi Aloka Medical Ltd, Japan) 30 min before the start of chemotherapy and 1 h after the end of the infusion. The analysis was performed using an automated software program (eTRACKING; Hitachi Aloka Medical Ltd, Japan), as previously described, and results were expressed as the percentage change from the baseline diameter. Doppler-derived flow measurements (a pulsed wave Doppler signal at a 70° angle) were also obtained continuously. The increase in blood flow after the release of the cuff was expressed as a percentage of the baseline flow. The endothelium-independent response to 25 μg of sublingual nitroglycerine (Nitorol spray, Eisai Co) was also calculated as the percentage change from the baseline diameter. Evaluation of lipid profiles In order to assess the lipid profiles, blood samples were collected in the morning on each day of chemotherapy after an overnight fast (indicated as “A” in Fig. 2). The plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and plasma glucose levels were
13
Int J Clin Oncol
measured enzymatically. The plasma low-density lipoprotein (LDL) cholesterol levels were calculated using Friedewald’s formula. Cell culture See the Supplemental Material. Measurement of the nitric oxide concentrations in the HUVECs See the Supplemental Material. Measurement of the PPARα DNA binding activity in the HUVECs See the Supplemental Material. Statistical analysis Statistical calculations were performed using the JMP statistical software package (SAS Institute, Cary, NC). An analysis of variance (ANOVA) was used for group comparisons. The data for the baseline patient characteristics and changes in lipid profiles are expressed as the mean ±SD. A paired t test was used to analyze differences between the values recorded at baseline and those obtained after the sixth course of TC treatment. Comparisons of the changes in %FMD and pentraxin-3 between three groups were made using the Wilcoxon signed-rank test as a non-parametric test. Correlations were assessed using the Spearman rank correlation coefficient. A p value
ORIGINAL ARTICLE
Fibrates protect against vascular endothelial dysfunction induced by paclitaxel and carboplatin chemotherapy for cancer patients: a pilot study Ayako Watanabe · Akiko Tanabe · Risa Maruoka · Kiyoko Nakamura · Koji Hatta · Yoshihiro J. Ono · Yoshito Terai · Masahide Ohmichi
Received: 29 May 2014 / Accepted: 10 December 2014 © Japan Society of Clinical Oncology 2014
Abstract Background Although we previously demonstrated that paclitaxel and carboplatin chemotherapy (TCchem) is associated with vascular toxicities, the underlying mechanisms remain unclear. Cisplatin is known to inhibit PPARα following microvascular damage to the kidneys. The primary aim of this study was to evaluate whether TCchem induces vascular endothelial dysfunction via systemic PPARα deficiency. In addition, human umbilical vein endothelial cells (HUVECs) were used to elucidate the mechanisms responsible for TCchem-induced vascular toxicities. Methods This study enrolled 45 gynecological cancer patients with normal lipid profiles who underwent surgical treatment followed by TCchem. The elevated triglyceride (TG) group included patients (n = 19) who exhibited hypertriglyceridemia during TCchem, and the stable TG group (n = 15) included patients with a normal TG level. Eleven patients exhibiting hypertriglyceridemia during TCchem were administered bezafibrate (fibrate group). Endothelial dysfunction was evaluated based on flowmediated dilation (FMD) values and serum pentraxin-3 levels measured before TCchem and immediately after the final TCchem. HUVECs were used to elucidate the biological mechanisms underlying the endothelial dysfunction induced by TCchem.
Electronic supplementary material The online version of this article (doi:10.1007/s10147-014-0779-y) contains supplementary material, which is available to authorized users. A. Watanabe · A. Tanabe (*) · R. Maruoka · K. Nakamura · K. Hatta · Y. J. Ono · Y. Terai · M. Ohmichi Department of Obstetrics and Gynecology, Osaka Medical College, 2‑7 Daigaku‑machi, Takatsuki, Osaka 569‑8686, Japan e-mail: [email protected]‑med.ac.jp
Results The administration of TCchem induced hypertriglyceridemia in 66 percent of the participants, and bezafibrate reduced the serum TG levels. Meanwhile, the decrease in flow-mediated dilatation (%FMD) induced by TCchem improved following treatment with bezafibrate. The serum pentraxin-3 level increased rapidly after TCchem and decreased following bezafibrate treatment. An in vitro examination demonstrated TCchem attenuated nitric oxide production and PPARα activity in HUVECs, which was partially improved by treatment with bezafibrate. Conclusion Bezafibrate prevents endothelial dysfunction induced by TCchem via TG-dependent and TG-independent mechanisms. Keywords Bezafibrate · Platinum-based chemotherapy · Carboplatin · Paclitaxel · Gynecological cancer · Triglyceride · FMD Abbreviations PtChem Platinum-based chemotherapy TG Triglycerides TCchem Paclitaxel and carboplatin combination chemotherapy FMD Flow-mediated dilation PPARα Peroxisome proliferator-activated receptor-α OC Ovarian cancer EC Endometrial cancer CVD Cardiovascular disease LDL Low-density lipoprotein HDL High-density lipoprotein hsCRP High-sensitivity C-reactive protein CDDP Cisplatin CBDCA Carboplatin PTX Paclitaxel
13
Fig. 1 Consort diagram explaining the recruitment process for each group. OC ovarian cancer, EC endometrial cancer, TG triglycerides, TC paclitaxel and carboplatin chemotherapy
Int J Clin Oncol
Screening Period (n=28) (OC n=14, EC n=14)
Intervenon Period (n=17) (OC n=9, EC n=8)
TC chemotherapy
Hypertriglyceridemia detected during TC Administraon of bezafibrate
Hypertriglyceridemia (OC n=10, EC n=9)
Elevated TG group (n=19) OC n=10 EC n=9
Introduction Cancer survivors are living longer after their initial diagnosis due to earlier diagnoses and improvements in treatment. Epithelial ovarian cancer (OC) and uterine endometrial cancer (EC) are major gynecological malignancies. Patients with these diseases generally undergo surgery, including bilateral oophorectomy, followed by the administration of platinum-based chemotherapy (PtChem). Although the acute adverse effects of combination chemotherapy have been well defined, recent research has focused on the late effects of PtChem. The long-term toxicity of chemotherapy is important due to the prolonged expected lifespan of these patients. Previous reports have suggested that PtChem is associated with vascular toxicities and serious vascular complications (e.g., myocardial infarction, stroke, and thromboembolic disease) [1–4] in testicular cancer survivors. Although the precise mechanisms remain unclear, several reports have demonstrated that cancer survivors who received chemotherapy showed increased plasma levels of endothelial and inflammatory markers, which might progress to atherosclerosis [5, 6], increased risks of hypertension [7–9], and dyslipidemia [7, 9]. We previously demonstrated that TCchem induces a significant increase in arterial stiffness 12 months after treatment and that the %FMD rapidly decreases after TCchem infusion [10]. Interestingly, our previous data demonstrated that serum triglyceride levels are significantly elevated in patients treated with TCchem. Portilla and colleagues reported a significant reduction in proximal tubule fatty acid oxidation leading to the accumulation of toxic fatty acid amphiphiles in patients with PtChem-induced acute renal failure [11–13]. A recent
13
Stable TG (OC n=4, EC n=5)
Stable TG (OC n=4, EC n=2)
Stable TG group (n=15) OC n=8 EC n=7
Fibrate group (n=11) OC n=5 EC n=6
study showed a threefold increase in the serum triglyceride levels on day 3 in mice treated with cisplatin, in addition to the accumulation of triglycerides in the urine and kidney tissue [14]. These metabolic changes and the degree of microvascular damage in the kidneys were improved by the administration of peroxisome proliferator-activated receptor-α (PPARα) ligands, including bezafibrate. These previous studies established that cisplatin deactivates PPARα in cisplatin-induced acute renal failure. There are currently no reports that have elucidated whether TCchem induces the development of systemic PPARα deficiency. Hence, the primary aim of the current pilot study was to evaluate whether TCchem induces vascular endothelial dysfunction via systemic PPARα deficiency. Furthermore, HUVECs were used to elucidate the mechanisms responsible for these effects based on in vitro examinations.
Methods Patients All patients evaluated in this study were under treatment at the Department of Obstetrics and Gynecology of Osaka Medical College Hospital. This study was a prospective study approved by the Institutional Review Board of Osaka Medical College. Written informed consent was obtained from all participants. The study population included 45 gynecological cancer patients who received adjuvant TCchem after surgical treatment according to established protocols between December 2006 and November 2012 (Fig. 1). We compared the following two periods: December 2006 to December 2009 as the screening
Int J Clin Oncol 0
Stable TG group (n=15) OC n=8 EC n=7 Elevated TG group (n=19) OC n=10 EC n=9
1
6
TC
Surgery
Fig. 2 Experimental design. OC ovarian cancer; EC endometrial cancer; TG triglycerides; TC paclitaxel and carboplatin combination chemotherapy; FMD flow-mediated dilation; after chemo: immediately after the final course of chemotherapy; A fasting blood sample collection
TC
TC
Fibrate group (n=11) OC n=5 EC n=6
Bezafibrate A FMD Blood Samples at baseline
period, and January 2010 to November 2012 as the intervention period. In the screening period, the patients in the surgery + TC group (n = 28) evaluated in our previous prospective study [10] were divided into the following two groups: the elevated TG group, including patients who exhibited hypertriglyceridemia (>150 mg/dl) under TCchem; and the stable TG group, including patients who exhibited a normal serum level of triglycerides (≤150 mg/ dl) under TCchem. In the intervention period, 17 patients were newly included. Blood samples were collected to monitor myelosuppression and fasting lipid profiles in the morning of each course of TCchem (indicated as ‘A’ in Fig. 2). In patients who exhibited hypertriglyceridemia (>150 mg/dl) twice during the study period, treatment with bezafibrate® (400 mg/day) was started and continued until the end of the planned adjuvant treatment period. Bezafibrate acts as a synthetic ligand for PPARα, which is mainly expressed in the liver, skeletal muscle, heart, and vascular endothelial cells [15]. The activation of PPARα results in plasma clearance of atherogenic triglyceride-rich lipoproteins [16, 17]. Participants who had received prior treatment for cancer and those with a history of cardiovascular disease or decompensated diabetes were excluded from this study. The exclusion criteria also included a history of clinically significant gastrointestinal, liver, or gallbladder disease, treatment with lipid- or glucose-lowering agents, and the use of hormone replacement therapy within the previous 1 year.
TC
A
A
A FMD Blood Samples at a er chemo
Treatment design A complete medical history was taken and a physical exam was performed in all patients. Pulmonary function tests and an electrocardiogram (ECG) were also performed, and the participants with abnormalities were excluded from this study. The patients underwent total hysterectomy, bilateral salpingo-oophorectomy, pelvic/para-aortic lymphadenectomy, and omentectomy, including the complete removal of all visible tumor tissue. TCchem was administered in all patients according to established protocols, after performing tumor staging using the International Federation of Gynecology and Obstetrics (FIGO) nomenclature. Briefly, patients were premedicated with dexamethasone (20 mg intravenously) for 30 min before the start of paclitaxel infusion. Both a histamine receptor type I blocker, diphenhydramine (50 mg orally), and a type II blocker, ranitidine (50 mg intravenously), were administered 30 min before paclitaxel infusion. Next, the patients received paclitaxel (175 mg/m2) intravenously for 3 h with monitoring of their vital signs (heart rate, blood pressure, pulse, and respiratory rate), followed by carboplatin (AUC 5 mg/ml) intravenously for 1 h on day 1 every 3 weeks for a total of six courses. The carboplatin dose was calculated using the Calvert formula: carboplatin dose (in mg) = AUC × (GFR + 25), where GFR is the glomerular filtration rate, estimated using the Jelliffe formula. Toxicity was assessed at each treatment cycle according to the National Cancer Institute Common Terminology
13
Criteria (version 3.0). Grade 3/4 hematologic toxicities associated with TCchem were managed with a dose reduction of carboplatin to an AUC of 4 or delay in administration for 7 days. Grade 2 or higher signs of peripheral neuropathy were managed with a dose reduction of paclitaxel to 75 %. If the above mentioned toxicities recurred after the first dose reduction or delay in the schedule, the carboplatin and paclitaxel dosages were again reduced to AUC = 3 and 75 %, respectively, or the schedule for chemotherapy was delayed. None of the patients in the current study required dose reduction, although 91.1 % of the patients were treated with a delay in the dosing schedule. All patients received a total of six courses of treatment. Study design The study design is shown in Fig. 2. %FMD and serum levels of pentraxin-3 and high-sensitivity C-reactive protein (hsCRP) were evaluated just before the first course of TCchem (baseline) and immediately after the final course of TCchem (after chemo). Details of the procedure used to measure the FMD values are described in the next paragraph. The levels of pentraxin-3 and hsCRP were measured using an enzyme-linked immunosorbent assay (ELISA) at the central clinical laboratory of Osaka Medical College. Measurement of the %FMD of the brachial artery In order to assess brachial %FMD, the right brachial artery diameter was measured on two-dimensional ultrasound images using a 7.0-MHz linear artery transducer and a ProSound SSD-Alpha 10 system (Hitachi Aloka Medical Ltd, Japan) 30 min before the start of chemotherapy and 1 h after the end of the infusion. The analysis was performed using an automated software program (eTRACKING; Hitachi Aloka Medical Ltd, Japan), as previously described, and results were expressed as the percentage change from the baseline diameter. Doppler-derived flow measurements (a pulsed wave Doppler signal at a 70° angle) were also obtained continuously. The increase in blood flow after the release of the cuff was expressed as a percentage of the baseline flow. The endothelium-independent response to 25 μg of sublingual nitroglycerine (Nitorol spray, Eisai Co) was also calculated as the percentage change from the baseline diameter. Evaluation of lipid profiles In order to assess the lipid profiles, blood samples were collected in the morning on each day of chemotherapy after an overnight fast (indicated as “A” in Fig. 2). The plasma total cholesterol, high-density lipoprotein (HDL) cholesterol, triglyceride, and plasma glucose levels were
13
Int J Clin Oncol
measured enzymatically. The plasma low-density lipoprotein (LDL) cholesterol levels were calculated using Friedewald’s formula. Cell culture See the Supplemental Material. Measurement of the nitric oxide concentrations in the HUVECs See the Supplemental Material. Measurement of the PPARα DNA binding activity in the HUVECs See the Supplemental Material. Statistical analysis Statistical calculations were performed using the JMP statistical software package (SAS Institute, Cary, NC). An analysis of variance (ANOVA) was used for group comparisons. The data for the baseline patient characteristics and changes in lipid profiles are expressed as the mean ±SD. A paired t test was used to analyze differences between the values recorded at baseline and those obtained after the sixth course of TC treatment. Comparisons of the changes in %FMD and pentraxin-3 between three groups were made using the Wilcoxon signed-rank test as a non-parametric test. Correlations were assessed using the Spearman rank correlation coefficient. A p value
