Intracellular uptake and cytotoxic effect in vitro of doxorubicin and epirubicin in human leukemic and normal hematopoietic cells

Summary Leukemic cells from patients presenting with acute nonlymphoblastic leukemia and normal hematopoietic bone marrow cells from healthy donors for allogeneic bone marrow transplantation were incubated for 3 h with doxorubicin and epirubicin at different concentrations. The intracellular uptake at the end of the incubation was determined by photofluorometry in leukemic cells from 15 patients and in normal cells from 9 donors for bone marrow transplantation. Cytotoxicity in vitro against granulocyte/macrophage colony-forming units (CFU-GM) was determined in normal cells from 7 donors, and in vitro toxicity against leukemic cells was determined by a clonogenic technique in cells from 6 patients and by vital dye staining (DiSC) following 4 days' culture in cells from 15 patients. Epirubicin was significantly less toxic than doxorubicin to normal hematopoetic cells (72%±20% survival of cells for epirubicin vs 45%±13% for doxorubicin at a concentration of 0.2 m;P0.005). As analyzed by the DiSC assay, 0.2 m epirubicin was slightly more toxic to leukemic cells than was the same concentration of doxorubicin (47% vs 61% survival,P0.01), but the clonogenic assay revealed no difference in toxicity to leukemic cells. At a concentration of 0.2 m, the mean intracellular uptake of epirubicin in leukemic cells was 0.43±0.26 nmol/mg protein as compared with 0.33±0.14 nmol/mg protein for doxorubicin (not significant). In normal cells, the uptake of epirubicin at a concentration of 0.2 m was 0.47±0.25 nmol/mg protein as compared with 0.31±0.21 nmol/mg protein for doxorubicin (not significant). The reduced myelotoxicity observed in vitro together with the retained toxicity to leukemic cells indicates that the therapeutic index of epirubicin is better than that of doxorubicin.     

Method for Dissection of Mesenteric Metastases in Mid-gut Carcinoid Tumors

With adequate medical management the midgut carcinoid tumor generally is an indolent malignancy associated with substantial life expectancy and appreciable life quality, even in the presence of liver metastases and significant tumor burden. Abdominal complications may occur in this entity of carcinoids owing to entrapment of intestines and encasement of mesenteric vessels by mesenteric metastases and associated marked mesenteric fibrosis. This may be the cause of abdominal pain, disabling diarrhea, weight loss to the extent of malnutrition, and eventually the risk of death with acute or chronic intestinal obstruction or intestinal gangrene. Operative removal of the mesentericointestinal lesion is often indicated to prevent or treat these complications but may be technically difficult when mesenteric metastases extend in the vicinity of major vessels in the mesenteric root. At laparotomy 56 patients with advanced midgut carcinoids underwent removal of the mesenteric tumor with a method for preserving the mesenteric vessels. This was feasible by mobilizing and releasing the right colon and mesenteric root from posterior adhesions, identifying the mesenteric artery below the pancreas, and free-dissecting this artery on the tumor capsule in the mobilized mesentery. Dissection was successful even with tumors initially judged inoperable unless tumor growth completely surrounded the mesenteric vessels or extended retroperitoneally. One patient was subjected to distal intestinal artery bypass. Symptom relief was been substantial and often of long duration after mesenteric tumor removal in patients who prior to surgery often had threatening intestinal ischemia. Patients with advanced midgut carcinoids may benefit markedly from dissectional removal of mesenteric tumors, which (conceivably better than conventional wedge resection) preserves the length of the remaining intestine.     

Spontaneous Reversal of P‐Glycoprotein Expression in Multidrug Resistant Cell Lines*

Abstract: Increased expression of P‐glycoprotein encoded by the mdr‐1 gene is a well‐characterised mechanism for resistance to cancer chemotherapeutic drugs in cell lines. However, the P‐glycoprotein expression after removal of the selection pressure has not fully been elucidated. The stability of P‐glycoprotein expression in the presence (+) and absence (?) of vincristine (30 or 150 nM) was studied in multidrug resistant K562 cell lines (VCR30+, VCR150+, VCR30? and VCR150?) for 11 months. The P‐glycoprotein protein and mdr‐1 mRNA levels were determined at regular intervals using flow cytometry and real‐time PCR, respectively. Chemosensitivity to a panel of antineoplastic drugs was measured using an MTT assay. The presence of vincristine (VCR30+ and VCR150+) resulted in high and stable levels of P‐glycoprotein and mdr‐1 mRNA during the whole period compared to wild type. As for the VCR30? and VCR150? subcultures, the expressions of P‐glycoprotein and mdr‐1 mRNA were stable for five months, and then the levels decreased rapidly. Concomitantly, the sensitivity to drugs known as P‐glycoprotein substrates was restored. In conclusion, resistant cells growing in the presence of the inducing drug have a stable P‐glycoprotein expression and resistance level, but removing the inducing drug may result in a sudden and rapid lowering of P‐glycoprotein and mdr‐1 mRNA levels as long as five months after drug withdrawal.     

3D conformal HDR brachytherapy and external beam irradiation combined with temporary androgen deprivation in the treatment of localized prostate cancer.

PURPOSE: To evaluate treatment outcome of 3D conformal high dose rate (HDR) brachytherapy and external beam irradiation (EBRT) combined with temporary androgen deprivation for patients with localized prostate cancer. PATIENTS AND METHODS: Between January 1997 and September 1999 we treated 102 patients with stage T1-3 N0 M0 prostate cancer. Stage T1-2 was found in 71, T3 in 31 patients. Median pretreatment PSA level was 15.3 ng/ml. After ultrasound-guided transrectal implantation of four afterloading needles, CT based 3D brachytherapy planning was performed. All patients received four HDR implants using a reference dose per implant of 5 or 7Gy. Time between each implant was 14 days. After brachytherapy EBRT followed up to 39.6 or 45.0 Gy. All patients received temporary androgen deprivation, starting 2-19 months before brachytherapy, ending 3 months after EBRT. RESULTS: Median follow-up was 2.6 years (range 2.0-4.1 years). Actuarial biochemical control rate was 87% at 2 years and 82% at 3 years. In 14 patients we noted biochemical failure, in five patients clinical failure. Overall survival was 90%, disease specific survival 98.0% at 3 years. Acute grade 3 toxicity occurred in 4%, late grade 3 toxicity in 5%. One patient developed a prostatourethral-rectal fistula as late grade 4 toxicity. The conformal quality of 300 HDR implants was analyzed using dose volume histograms. CONCLUSIONS: 3D conformal HDR brachytherapy and EBRT combined with temporary androgen deprivation is an effective treatment modality for prostate cancer with minimal associated toxicity and encouraging biochemical control rates after a median follow-up of 2.6 years.     

Infection with oncolytic herpes simplex virus-1 induces apoptosis in neighboring human cancer cells: a potential target to increase anticancer activity.

PURPOSE: The antitumor efficacy of a herpes simplex virus (HSV)-1 oncolytic virus depends on the cytotoxic effect of the virus, but also on viral replication and spread within the tumor. Apoptosis is considered a defense mechanism of infected cells that minimizes the spread of viral progeny by limiting cellular production of virus. We sought to determine whether oncolytic HSV-1 infection induces apoptosis in neighboring, uninfected cells and whether manipulation of apoptosis can increase viral replication and cytotoxicity. EXPERIMENTAL DESIGN: NV1066 is an oncolytic HSV-1 mutant that contains the marker gene for enhanced green fluorescent protein. OCUM human gastric cancer cells were infected with NV1066 in vitro and inspected for apoptosis by Hoechst and terminal deoxynucleotidyltransferase-mediated nick end labeling staining and for infection by expression of green fluorescence. RESULTS: A significant increase in apoptosis was seen in cells infected by NV1066. More interestingly, a significant percentage (10%) of uninfected cells also proceeded to apoptosis. After NV1066 infection, cells were also treated with N-acetylcysteine (NAC), an inhibitor of apoptosis. By day 4 after infection, 2.7x more NV1066 was produced in cells exposed to NAC than in those not exposed to NV1066 (P = 0.04). NAC also increased tumor kill when administered with virus. CONCLUSIONS: These data suggest that NV1066 induces apoptosis in uninfected cocultured cells, potentially hindering propagation of viral progeny and concomitant tumor kill. Inhibition of apoptosis may improve the efficacy of oncolytic HSV-1 therapy.     

Genetic analysis of the RNASEL gene in hereditary, familial, and sporadic prostate cancer.

PURPOSE: The RNASEL gene has been proposed as a candidate gene for the HPC1 locus through a positional cloning and candidate gene approach. Cosegregation between the truncating mutation E265X and disease in a hereditary prostate cancer (HPC) family and association between prostate cancer risk and the common missense variant R462Q has been reported. To additionally evaluate the possible role of RNASEL in susceptibility to prostate cancer risk, we performed a comprehensive genetic analysis of sequence variants in RNASEL in the Swedish population. EXPERIMENTAL DESIGN: Using 1624 prostate cancer cases and 801 unaffected controls, the truncating mutation E265X and five common sequence variants, including the two missense mutations R462Q and D541E, were evaluated for association between genotypes/haplotypes and prostate cancer risk. RESULTS: The prevalence of E265X carriers among unaffected controls and prostate cancer patients was almost identical (1.9 and 1.8% in controls and cases, respectively), and evidence for segregation of E265X with disease was not observed within any HPC family. Overall, the analyses of common sequence variants provided limited evidence for association with prostate cancer risk. We found a marginally significant inverse association between the missense mutation D541E and sporadic prostate cancer risk (odds ratio, 0.77; 95% confidence interval, 0.59-1.00) and reduced risk of prostate cancer in carriers of two different haplotypes being completely discordant. CONCLUSIONS: Considering the high quality in genotyping and the size of this study, these results provide solid evidence against a major role of RNASEL in prostate cancer etiology in Sweden.     

Immunomagnetic enrichment, genomic characterization, and prognostic impact of circulating melanoma cells.

PURPOSE: The finding of melanoma cells in the peripheral blood, thus far mainly inferred from the PCR-based demonstration of tyrosinase mRNA, has been associated with metastatic melanoma. Neither the malignant nature nor the prognostic significance of circulating cells could be established. To address this question, we analyzed immunomagnetically isolated circulating melanoma cells for chromosomal aberrations and performed a clinical follow-up study of the enrolled patients. EXPERIMENTAL DESIGN: In a prospective study, blood samples were taken from 164 melanoma patients and 50 donors without malignant disease. Circulating melanoma cells were enriched by immunomagnetic cell sorting using a murine monoclonal antibody against the melanoma-associated chondroitin sulfate proteoglycan. To prove the malignant origin of the positive cells and to define their chromosomal aberrations, we analyzed the genomes of 15 individually isolated cells from seven patients by single-cell comparative genomic hybridization (SCOMP). RESULTS: Absolute and relative frequencies of circulating melanoma cells were associated with stage and with the presence or absence of detectable tumor. The detection of two or more cells correlated significantly with a reduced survival of patients with metastatic melanoma. All of the cells that were analyzed by SCOMP displayed multiple chromosomal changes and carried aberrations typical for melanoma. CONCLUSIONS: Immunomagnetic enrichment enables isolation and genomic characterization of circulating melanoma cells. The prognostic impact on survival of metastatic patients apparently reflects the aggressiveness of an ongoing tumor spread. Direct genomic analysis of the enriched and isolated cells will help to clarify the molecular-genetic basis of the establishment of generalized melanoma.     

Human kallikrein 6 (hK6): a new potential serum biomarker for diagnosis and prognosis of ovarian carcinoma.

PURPOSE: The discovery of new ovarian cancer biomarkers that are suitable for early disease diagnosis and prognosis may ultimately lead to improved patient management and outcomes. PATIENTS AND METHODS: We measured, by immunoassay, human kallikrein 6 (hK6) concentration in serum of 97 apparently healthy women, 141 women with benign abdominal diseases, and 146 women with histologically proven primary ovarian carcinoma. We then calculated the diagnostic sensitivity and specificity of this test and examined the association of serum hK6 concentration with various clinicopathologic variables and patient survival. RESULTS: Serum hK6 concentration between normal and benign disease patients was not different (mean, 2.9 and 3.1 micro g/L, respectively). However, hK6 in presurgical serum of ovarian cancer patients was highly elevated (mean, 6.8 micro g/L; P <.001). Serum hK6 decreased after surgery (to a mean of 3.9 micro g/L) in 68% of patients. The diagnostic sensitivity of serum hK6 at 90% and 95% specificity is 52% and 47%, respectively, in the whole patient population. For early stage disease (stage I or II), sensitivity is approximately 21% to 26%. When combined with CA-125, at 90% specificity, sensitivity increases to 72% (for all patients) and to 42% in stage I or II disease. Serum hK6 concentration correlates moderately with CA-125 and is higher in patients with late-stage, higher-grade disease and in patients with serous histotype. Preoperative serum hK6 concentration is a powerful predictor of disease-free and overall survival in both univariate and multivariate analyses. CONCLUSIONS: Serum hK6 concentration seems to be a new biomarker for ovarian carcinoma and may have value for disease diagnosis and prognosis.