Endometrial epithelial-mesenchymal transition (EMT) by menstruation-related inflammatory factors during hypoxia.

Endometriosis is characterised by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal haemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, hypoxia is a well-known inducer of fibrosis that may be associated with epithelial-mesenchymal transition (EMT). However, the complex molecular interactions between hypoxia and proinflammatory menstruation-related factors, PGE2 and thrombin, a PAR1 agonist, on EMT in endometriosis have not been fully characterised. To explore the effects of hypoxia and proinflammatory factors on EMT-like changes in endometrial cells, we determined the effects of PGE2 and thrombin (P/T) on EMT marker expression and cell migration in three dimensional cultured human endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Treatment of EECs with P/T under hypoxia stimulated cell migration, increased the expression of mesenchymal N-cadherin, vimentin and C-X-C chemokine receptor type 4 (CXCR4), and reduced the expression of epithelial E-cadherin. Furthermore, treatment with C-X-C motif chemokine ligand 12 (CXCL12), a ligand for CXCR4, increased EMT marker expression and cell migration. In ESCs, P/T or oestrogen treatment under hypoxic conditions increased the expression and secretion of CXCL12. Taken together, our data show that hypoxic and proinflammatory stimuli induce EMT, cell migration and inflammation in EECs, which was increased by CXCL12 derived from ESCs. These data imply that inflammatory mediators in retrograde menstrual fluid contribute to ectopic endometrial EMT and migration in the presence of peritoneal hypoxia.

Hydrothermal Ethanol Flames in Co-Flow Jets.

Results on the autoignition and stabilization of ethanol hydrothermal fames in a Supercritical Water Oxidation (SCWO) reactor operating at constant pressure are reported. The flames are observed as luminous reaction zones occurring in supercritical water; i.e., water at conditions above its critical point (approximately 22 MPa and 374 °C). A co-flow injector is used to inject fuel (inner flow), comprising an aqueous solution ranging from 20 %-v to 50 %-v ethanol, and air (annular flow) into a reactor filled with supercritical water at approximately 24.3 MPa and 425 °C. Results show hydrothermal fames are autoignited and form diffusion flames which exhibit laminar and/or turbulent features depending upon flow conditions. Two orthogonal camera views are used; one providing a backlit shadowgraphic image of the co-flow jet and the other providing color images of the flame. In addition, spectroscopic measurements of flame emissions in the UV and visible spectrum are discussed.

Characterization of cerebral salt wasting after subarachnoid hemorrhage model induced by endovascular puncture.

Cerebral salt wasting (CSW) frequently occurs concomitantly with an aneurysmal subarachnoid hemorrhage (SAH). CSW induces excessive natriuresis and osmotic diuresis, and reduces the total volume of blood. We previously reported that a rat model with SAH induced by endovascular puncture (EP) exhibited CSW. Therefore, we investigated the relationship between the spread of bleeding in the subarachnoid space and the intensity of CSW. We also investigated the development of CSW in different SAH models. SAH was induced by EP or by 0.3 mL of blood injection (BI) into the cisterna magna. To evaluate the occurrence of CSW, urine was cumulatively collected at the onset of SAH to 6 h later and analyzed for sodium (Na) excretion. SAH was classified from grade 1 (no bleeding) to grade 4 (severe bleeding) based on the spread of bleeding in the subarachnoid space. In the EP model (SAH grade > 2) as the SAH grade increased, the volume of urine and Na excretion also significantly increased. Although the BI model rats exhibited SAH of grade 4, the volume of urine and Na excretion did not change. Therefore, our conclusion is that the spread of bleeding in the subarachnoid space may not cause CSW.

Inhibitory effect of hydrocortisone on cerebral salt wasting after subarachnoid hemorrhage in rats.

Cerebral salt wasting (CSW) frequently occurs concomitantly with subarachnoid hemorrhage (SAH). CSW induces excessive natriuresis and osmotic diuresis, reduces total blood volume, aggravates cerebral vasospasm and causes cerebral ischemia after SAH. This study examined the inhibitory effect of hydrocortisone on CSW in rat SAH models. Hydrocortisone had an inhibitory effect on CSW because hydrocortisone functioned in a dose-dependent manner to inhibit the increase in sodium excretion and sodium/potassium ratio after SAH onset. We conclude that hydrocortisone is a useful drug for the treatment of CSW after SAH.

New approach for chronic renal failure model by direct kidney injection of doxorubicin in rats.

Several experimental chronic renal failure (CRF) models are available for testing new drugs. A CRF model induced by the intravenous injection of 2 mg/kg of doxorubicin (DXR) twice during a 20-day interval reportedly results in pathological characteristics similar to glomerular sclerosis seen clinically. However, it normally takes more than 16 weeks to create this CRF model. We used three methods of direct drug injection into the kidney of rats to determine the method that would induce CRF within 4 weeks; Method A: DXR was injected directly into both kidneys; Method B: DXR was injected directly into the left kidney immediately after right nephrectomy; Method C: DXR was injected directly into the left kidney 1 week before right nephrectomy, and DXR was injected again directly into the left kidney. As a result, urinary protein, blood urea nitrogen (BUN), creatinine and creatinine clearance were significantly changed >1 week after the injection of DXR by Method C. Quantification of tissue transforming growth factor-beta1 (TGF-beta1), which is a prime fibrogenic cytokine in renal fibrosis, significantly increased in the kidney. A light microscopic image showed glomerular decrement, tubular dilation and atrophy and vacuolation of parenchyma. In conclusion, the results of this study demonstrate that the DXR model using Method C develops CRF within 4 weeks.

Experimental model for investigating hyponatremia after subarachnoid hemorrhage in rats.

Hyponatremia is a common complication in patients with aneurysmal subarachnoid hemorrhage (SAH). Such patient demonstrates excessive natriuresis and an increased risk of symptomatic cerebral vasospasm. However, the precise mechanisms underlying SAH induced hyponatremia remain unclear. In the present study, in order to establish an experimental model of hyponatremia following SAH, we induced SAH in rats, and evaluated the serum sodium (Na) levels, Na excretion and physiological parameters. Twenty-four male Wistar rats were used. SAH was induced by an endovascular puncture method. The mean arterial pressure (MAP), intracranial pressure (ICP), and cerebral blood flow (CBF) were monitored continuously. The urine was collected cumulatively for 12 hours after SAH, and the urine Na concentration was determined with a spectrophotometer. The serum Na levels were measured at 12 hrs, 2 and 4 days following the SAH induction. The mean (+/- standard deviation) baseline ICP was 3.5 +/- 2.6 mmHg, and increased to 67.4 +/- 17.6 mmHg immediately following induction of SAH. CBF decreased rapidly, and then gradually recovered to 70-80% of baseline. The urine volume and total Na excretion were significantly increased in comparison to those of the sham (P < 0.05). The serum Na level was significantly decreased at 4 days following SAH (P < 0.05). The present results demonstrated for the first time that rats with SAH exhibited excessive natriuresis. The endovascular puncture model is suitable for investigating hyponatremia that occurs concomitantly with natriuresis and diuresis after SAH.

Recent trends in pediatric fluid therapy.

Virtually all hospitalized pediatric patients require some form of intravenous fluid administration. The foundation of current pediatric fluid therapy practice was formulated in the 1950s when pediatricians were dealing with relatively simple dehydration and normal homeostasis could largely be assumed. Recent advances in pediatric medicine have resulted in increased severity of illness and normal physiology can no longer be assumed. The traditional approach to pediatric fluid therapy has been recently challenged by the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), cerebral salt wasting syndrome (CSWS), diabetic ketoacidosis (DKA) and hyponatremia caused by the inappropriate use of hypotonic solutions, all of which involve unusual sodium and serum osmolarity dynamics causing life threatening central nervous system (CNS) pathophysiology. In this review, we give an overview of the recent understanding of pediatric fluid therapy. The widespread use of acetate in place of lactate as a bicarbonate precursor and the expanding role of nonalbumin plasma expanders in pediatrics are also discussed as they will play a clinical role in the near future.

Metabolic properties of the acid and lactone forms of HMG-CoA reductase inhibitors.

To gain a better understanding of the metabolic properties between the open acid and lactone form of HMG-CoA reductase inhibitors (statins), the paper focused primarily on characterizing the metabolic properties of statins. We compared the metabolism of the acid and lactone forms of several statins, including atrovastatin, simvastatin, cerivastatin fluvastatin, pitavastatin and rosuvastatin with respect to metabolic clearance, CYP enzymes involved and drug-drug interactions. A remarkable increase in metabolic clearance was noted for all lactones compared with all acids except for pitavastatin lactone. The metabolic clearances of the atrovastatin, simvastatin, cerivastatin, fluvastatin and rosuvastatin lactones were 73-, 70-, 30-, 7- and 64-fold higher, respectively, than those of the corresponding acids. CYP2Cs were critically involved in the metabolism of cerivastatin, fluvastatin and pitavastatin acids. In contrast, CYP2Cs were not involved in the metabolism of the corresponding lactones and CYP3A4 was mainly involved. Moreover, a substantial difference in the metabolic inhibition of statins was found between acids and lactones. Overall, the study demonstrates that CYP-mediated metabolism of lactones is also a common metabolic pathway for statins and that the CYP3A4-mediated metabolism of the lactone forms clearly will need to be taken into account in assessing mechanistic aspects of drug-drug interaction involving statins.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase: similarities and difference in the metabolism of pitavastatin in monkeys and humans.

1. To elucidate any potential species differences, the in vitro metabolism of pitavastatin and its lactone was studied with hepatic and renal microsomes from rats, dogs, rabbits, monkeys and humans. 2. With the addition of UDP-glucuronic acid to hepatic microsomes, pitavastatin lactone was identified as the main metabolite in several animals, including humans. 3. Metabolic clearances of pitavastatin and its lactone in monkey hepatic microsome were much greater than in humans. 4. M4, a metabolite of pitavastatin with a 3-dehydroxy structure, was converted to its lactone form in monkey hepatic microsomes in the presence of UDP-glucuronic acid as well as to pitavastatin. These results implied that lactonization is a common pathway for drugs such as 5-hydroxy pentanoic acid derivatives. 5. The acid forms were metabolized to their lactone forms because of their structural characteristics. 6. UDP-glucuronosyltransferase is the key enzyme responsible for the lactonization of pitavastatin, and overall metabolism is different compared with humans owing to the extensive oxidative metabolism of pitavastatin and its lactone in monkey.

Metabolic fate of pitavastatin, a new inhibitor of HMG-CoA reductase: human UDP-glucuronosyltransferase enzymes involved in lactonization.

1. Pitavastatin is a potent competitive inhibitor of HMG-CoA reductase little metabolized in hepatic microsomes. Pitavastatin lactone, which can be converted back to the unchanged form, is the major metabolite of pitavastatin in humans. To clarify the mechanism of the lactonization of pitavastatin and the metabolic properties of the lactone, we performed experiments in vitro. 2. On addition of UDP-glucuronic acid, human hepatic microsomes produced pitavastatin lactone and an unknown metabolite (UM-2). UM-2 was converted to its unchanged form by enzymatic hydrolysis and to a lactone form non-enzymatically. Using several human UGT-expressing microsomes, UGT1A3 and UGT2B7 were principally responsible for glucuronidation of pitavastatin leading to lactonization. 3. No marked difference in intrinsic clearance between pitavastatin and its lactone form was detected in human hepatic microsomes. 4. Pitavastatin lactone showed no inhibitory effects on CYP2C9- and CYP3A4-mediated metabolism of model substrates in contrast to other HMG-CoA reductase inhibitors. 5. The mechanism of pitavastatin lactone formation has been clarified, in that glucuronidation by UGT occurs first followed by lactonization via an elimination reaction. It was also found that pitavastatin lactone demonstrates no drug-drug interactions.

Toxicological study of a new maintenance fluid, Veen 3G, in rats.

A study of the different volume and infusion rates of a new maintenance fluid, Veen 3G, on the general conditions of rats was investigated during the 14 days after infusion. In Experiment I, 100 ml/kg and 200 ml/kg of Veen 3G were infused at a rate of 300 ml/kg/h in male and female rats. Results were compared with those for Gurunon Ringer solution (GRS) in male and female rats. We observed only transient polyuria in animals administered by each dose of Veen 3G and GRS for 0-15 min after infusion. Necropsy was not observed in any of the animals tested 14 days after infusion. In Experiment II, 200 ml/kg of Veen 3G was infused at rates of 200, 400, 800 and 1600 ml/kg/h in male rats. At 800 and 1600 ml/kg/h, irregular respiration and decrease in movement were observed concomitantly with polyuria. Three out of 4 rats died immediately after the infusion of Veen 3G at a rate of 1600 ml/kg/h, and one rat was still alive 14 days after the infusion. In this experiment, 200 ml/kg Veen 3G was safe when we infused at a rate of less than 400 ml/kg/h in male rats. Since this rate is about 27-80 times higher than that used clinically in maintenance treatment, Veen 3G is suggested to be safe, with the exception of polyuria, in clinical situations at the standard infusion rate (5-15 ml/kg/h).

Infusion of maintenance fluids with glucose (Veen 3G) is superior to maltose infusion (Actit) in the rate of energy utilization in rabbits.

In the present study, we examined the rates of urinary excretion of glucose and maltose after an infusion of maintenance fluid with glucose or maltose in adult rabbits. Three maintenance fluids (sugar-free, 5% glucose [Veen 3G] and 5% maltose [Actit]), which contained different sugars but were identical in electrolyte and acetate compositions and concentrations (Na: 45, K: 17, Mg: 5, Cl: 37, H2PO4: 10 and CH3COO: 20 mEq/l), were used in this study. In addition, the optimum infusion speed for maintenance therapy (10 ml/kg/h) was used. Animals were not given food or water during the 10-day period of administration. The body weights of the animals were measured every day. The concentrations of total protein, albumin, free fatty acids and glucose in the serum were measured. Urine samples for determination of glucose and maltose concentrations were collected from the 1st to 10th administrations. After infusion with 5% maltose, urinary maltose excretion decreased time-dependently, while that of glucose increased. This suggests that maltase activity time-dependently increases after infusion with maltose. In addition, total sugar was only minimally excreted into urine in the 5% glucose group compared with the 5% maltose group. Thus, the glucose infusion was superior to the maltose infusion in the rate of energy utilization. However, neither the loss of body weight nor the increase in concentration of free fatty acids in serum differed significantly among the 3 groups. In conclusion, infusion of maintenance fluid with 5% maltose results in the excretion of maltose and glucose into urine, since enzymatic hydrolysis of maltose to glucose is limited to that by maltase.

Toxicological study of a glucose-added acetic acid maintenance infusion solution (VEEN 3G Inj.) local irritation test.

The local irritating effect of Veen 3G Inj. (glucose-added acetic acid maintenance infusion solution) was examined in male rabbits. We studied the local irritating effect of the infusion solution compared with that of Ringer's solution, 5% sulfobromophthalein sodium injection, distilled water for injection or glucose-added Ringer's solution. In the vascular irritation test, macroscopical and histopathological changes induced by the infusion solution were not observed in the vessels. Moreover, in the hemolytic test, hemolysis of rabbit erythrocyte was not observed in the mixture with the infusion solution. In the present study, no change suggesting irritation by the infusion solution was observed in the in vivo vascular irritation test using the auricular vein of rabbits or in the in vitro hemolytic test using rabbit erythrocyte. In conclusion, in clinical use the infusion solution produces extremely slight adverse effects, such as vessel pain and phlebitis on the injection site.

Pathophysiological relevance of the CD14 receptor in surgical patients: biological activity of endotoxin is regulated by the CD14 receptor.

Endotoxins (lipopolysaccharides, LPSs) are potent bacterial poisons, and they are always present in the intestine in considerable numbers. Stress, such that as a resulting from multiple injuries, burns, hypovolemia, hypoxia, intestinal ischemia, and surgery can lead to a breakdown of the gut barrier, allowing endotoxins to enter the systemic circulation via translocation. However, estimating the biological activity of translocated circulating endotoxins and identification of the mechanisms regulating their biological activities remain complex problems. CD14 has been found to exist as a soluble protein in the serum and as a glycosylphosphatidylinositol (GPI)-anchored protein of myeloid lineage cells. It plays key roles in both LPS-induced activation and in LPS internalization by cells. In this article, we outline: (i) the biological activity of circulating endotoxin; and (ii) the role of membrane and/or soluble CD14 regulating the bioactivity of circulating endotoxin in a human model of postoperative endotoxemia.

Significance of lymphoscintigraphic mapping with Tc-99m human serum albumin and tin colloid in sentinel lymph node biopsy in breast cancer.

Sentinel lymph node biopsy (SLNB) in breast cancer is considered in order to spare node-negative patients from axillary lymph node dissection. To assess the clinical significance of lymphoscintigraphic mapping in SLNB, we analyzed the lymphatic drain to the sentinel lymph nodes (SLNs) in terms of the pattern and direction of the hot spot. Twenty-three breast cancer patients were enrolled for SLNB. Before surgery, lymphoscintigraphic mapping of SLN was performed using Tc-99m human serum albumin (HSA) and tin colloids, and the hot spot was marked. The Tc-99m HSA and tin colloids were subcutaneously injected above the tumor and peritumor sites, respectively, and lymphoscintigraphic scanning was monitored every 5 to 10 min, for up to 2 h after injection. The SLN was identified using a combination of a blue dye, indigocalmine, and a gamma probe during surgery. The hot spot pattern and direction of the lymphatic drains were evaluated in 21 of 23 cases. Two cases did not have a hot spot. Single, double, and multiple hot spots were observed in 12 cases (52.1%), 8 cases (34.7%), and 1 case (4.3%), respectively. The positions of the hot spots were: axillary (n=17, 80.9%), axillary and sternal (n=3, 14.2%), and phrenic (n=1, 4.7%). The sensitivity and specificity rates in SLNB were 66.6% and 100%, respectively, and the overall predictive rate was 85.7%. Lymphoscintigraphy produced false negatives in three cases (33.3%), including one on the phrenic side. Lymphoscintigraphic mapping with Tc-99m HSA and tin colloids is useful for determining the SLN, and avoiding a false negative. The pattern and direction of the lymphatic drain to the SLN in scintigraphy need to be considered for the elimination of axillary lymph node dissection in node-negative patients with breast cancer.

Toxicity of theophylline depends on plasma concentration by single and also repeated dosing in rats.

The purpose of this study was to evaluate the relationship between the in vivo toxicity and plasma concentration of theophylline. Theophylline was administered intravenously in single doses ( 50, 100, 150 and 200 mg kg(-1)once a day) or repeated doses (12.5, 25 and 90 mg kg(-1)/day for 28 days) in rats. Plasma concentrations of theophylline increased dose-dependently in both single and repeated doses, and there were no differences due to effects of 28-times repeated administration. Neither single dose at 50 mg kg(-1)nor repeated dose at 12.5 mg kg(-1)/day injections of theophylline showed toxic signs, in which plasma concentrations of theophylline were less than 110 and 22.5 microg ml(-1), respectively. Theophylline induced myocardial fibrosis in 25 mg kg(-1)/day and more treated groups: in which plasma concentrations of theophylline were more than 50 microg ml(-1). At doses of 100 mg kg(-1)(single) and 90 mg kg(-1)/day (repeated), theophylline caused tachypnea and excitement of movement. Each theophylline concentration in plasma was more than 194 microg ml(-1)in single 100 mg kg(-1)and 162 microg ml(-1)in repeated 90 mg kg(-1)/day injections, respectively. Death was observed at a dose of 200 mg kg(-1), in which the plasma concentration of theophylline was more than 264 microg ml(-1). Moreover, the recovery period from signs of toxic poisoning to normality in the 200 mg kg(-1)treated group was greater than that in the 150 mg kg(-1)and less treated groups. The results indicated that the in vivo toxicity of theophylline is highly dependent on plasma concentrations in rats which received single and also repeated doses of theophylline.

An analysis of relapsed breast cancer in patients previously treated with breast conserving surgery.

BACKGROUND: Recent advances in breast surgery have focused on breast conserving surgery in combination with radiotherapy. In the present study, we examine by retrospective analysis 105 patients with breast cancer who received breast conserving surgery for factors influencing disease free survival. METHODS: The analysis was performed on 105 patients with breast cancer who received breast conserving surgery in our department, including 38 patients without radiotherapy and 67 patients treated with radiotherapy. The disease-free survival of the patients was analyzed using the Kaplan-Meier method. The relapsed patients were assessed by examining pathological features and gene expression by immunohistochemical staining. RESULTS: There was no significant difference in the disease free survival at 5 years between patients without radiotherapy (89.6%) and with radiotherapy (94.5%). Relapse after breast conserving surgery was found in 6 patients including 4 patients without radiotherapy and 2 patients with radiotherapy. Local relapse and bone metastasis were found in 4 (3.8%) and 2 patients, respectively. Among the 4 local relapses, 1 patient had received radiotherapy and 3 patients had not. There was no significant difference between the type of relapse in terms of lymph node metastasis, hormone receptor, nuclear grade and intraductal component, but more vessel invasion was observed in the 2 cases with bone metastasis. The overexpression of apoptosis and angiogenesis genes such as p53, Bax, Bcl-XL, Bcl-2 and VEGF was not common in the relapsed patients, whereas the overexpression of drug resistance genes, either P-gp or MRP1, was found in the all patients. CONCLUSIONS: Although radiotherapy may reduce the incidence of local relapse and increase disease free survival after breast conserving surgery, the development of an effective adjuvant chemotherapy based on drug resistance markers, is also required.

Inhomogeneity in the appearance of electrical remodeling during chronic rapid atrial pacing: evaluation of the dispersion of atrial effective refractoriness.

In the present study, the long-term process of progression of electrical remodeling at various atrial sites, which is not well understood, was compared while monitoring continuously the electrophysiologic parameters at multirecording sites in canine atria during continuous atrial burst pacing. A rapid pacing device was implanted in 5 dogs, and continuous atrial burst pacing (400 beats/min) was delivered at the right atrial appendage (RAA). Four pairs of epicardial wire electrodes were sutured on (1) the RAA, (2) Bachmann's bundle (BB), (3) the right atrium close to the inferior vena cava (IVC), and (4) the left atrium (LA). The distal ends of those wires were exteriorized posteriorly and used for pacing and recording. The atrial effective refractory period (AERP), AERP dispersion (AERPd), atrial conduction time (CT) and inducibility of atrial fibrillation (AF) were evaluated during burst pacing for 14 days and during the subsequent 7 days' recovery. The AERP at the LA pacing site was shorter than that at the other sites on day 0. The AERP shortening was greater in the RAA and LA sites than in the BB and IVC sites. The AERPd increased during pacing and reached the maximum level on day 3, and then decreased during the recovery phase. Prolongation of CT tended to be longer between the RAAand IVC sites than that between the other sites. The incidence of AF induction became higher in accordance with the time course of the rapid pacing phase. There was another peak of AF induction on days 7-10. In a canine chronic rapid atrial stimulation model, the progression of electrical remodeling (ie, the shortening of the AERP and the prolongation of the CT) was not homogeneous in both atria, the AERPd showed a temporal increase between days 3 and 7 and matched the increase in AF inducibility at the LA pacing site, the increase in the AERPd was mainly caused by more rapid AERP shortening at the RAA or LA sites, and the LA site always showed a shorter AERP than the other atrial sites in the control state and during the rapid pacing phase, whereas AF inducibility was higher at the LA site than the other sites.

A phase II trial of mitomycin C, 5'-deoxy-5-fluorouridine, etoposide and medroxyprogesterone acetate (McVD-MPA) as a salvage chemotherapy to anthracycline-resistant tumor in relapsed breast cancer and its mechanism(s) of antitumor action.

To assess the therapeutic efficacy in the combination of mitomycin C (MMC), 5'-deoxy-5-fluorouridine (5'-DFUR), etoposide (VP-16) and medroxyprogesterone acetate (MPA) (McVD-MPA) to anthracycline-resistant tumor as a salvage chemotherapy, a phase II trial was conducted in patients with relapsed breast cancer. Fifty-five patients were enrolled in this trial and 54 were assessable, who had all previously been treated with an anthracycline regimen. The treatment schedule was designed with the intravenous administration of MMC (6 mg/m2) on day 1 followed by peroral administration of VP-16 (75 mg/m2) on day 2, 4, 6 and the peroral administration of 5'-DFUR (600 mg/m2) and MPA (400 mg/m2) on day 1 through 21 in one cycle. The overall tumor response rate was 40.7% (22/54) including 16.6% (9 cases) in complete response and 24.0% (13 cases) in partial response, and the long no change (NC) was observed in 18.5% (10/54) out of 44.4% (24/54) in NC. Of the patients with primary resistance to anthracycline 30.0% responded to McVD-MPA therapy. Bone and liver metastases responded in 50.0% and 50.0%, whereas soft tissue and lung metastases responded in 36.8% and 35.2%, respectively. The mean time to response and response duration were 2.7 and 15.6 months, respectively. The overall survival of the patient treated with the McVD-MPA was superior to the non-treatment of second line therapy, and the median survival between McVD-MPA and non-treatment was 86 days and 50 days, respectively. The major adverse effect was observed in hematological toxicity (31.7%) such as leukopenia and thrombocytopenia and non-hematological toxicity of gastrointestinal events (31.7%), the toxicity was less than grade 2, and was tolerable during the treatment. In the experiment of MDA-MB-231 breast cancer cell line that was overexpressed with P-glycoprotein (P-gp) and multidrug resistance associated protein (MRP), the mechanism(s) by which McVD-MPA induces the antitumor effect to anthracycline-resistant tumor may be explained at least in part as follows: i) The treatment of MMC suppressed the expression of P-gp and MRP in a dose-and time-dependent manner, connecting the increase of the intracellular concentration of VP-16; ii) The treatment of MMC enhanced the expression of thymidine phosphorylase to increase the production of 5-FU from 5'-DFUR in the antiangiogenic effect of MPA. These results indicate that the combination chemotherapy of the McVD-MPA may be an effective regimen to anthracycline-resistant tumor as a salvage chemotherapy to prolong the survival in the patient with relapsed breast cancer.