Mitochondria organelle transplantation: introduction of normal epithelial mitochondria into human cancer cells inhibits proliferation and increases drug sensitivity.

Mitochondrial dysfunction of cancer cells includes increased aerobic glycolysis, elevated levels of ROS, decreased apoptosis, and resistance to chemotherapeutic agents. We hypothesized that the introduction of normal mitochondria into cancer cells might restore mitochondrial function and inhibit cancer cell growth, and reverse chemoresistance. First, in the present study, we tested if mitochondria of immortalized, untransformed mammary epithelial MCF-12A cells could enter into human cancer cell lines. Second, if introducing normal mitochondria into cancer cells would inhibit proliferation. And third, would the addition of normal mitochondria increase the sensitivity of human breast cancer MCF-7 cells to chemotherapy. We found that JC-1-stained mitochondria of immortalized, untransformed mammary epithelial MCF-12A cells can enter into the cancer cell lines MCF-7, MDA-MB-231, and NCI/ADR-Res, but cannot enter immortalized, untransformed MCF-12A cells. The normal mitochondria from immortalized, untransformed MCF-12A cells suppressed the proliferation of MCF-7 and NCI/ADR-Res cells in a dose-dependent pattern, but did not affect the proliferation of immortalized, untransformed MCF-12A cells. The normal mitochondria from immortalized, untransformed MCF-12A cells increased the sensitivity of human breast cancer MCF-7 cells to doxorubicin, Abraxane, and carboplatin. In conclusion, the introduction of normal mammary mitochondria into human breast cancer cells inhibits cancer cell proliferation and increases the sensitivity of the MCF-7 human breast cancer cell line to doxorubicin, Abraxane, and carboplatin. These results support the role of mitochondrial dysfunction in cancer and suggest the possible use of targeted mitochondria for cancer therapeutics.

Severity of canine myocardial infarcts in relation to indices of oxygen demand: preservation of myocardial creatine kinase activity by vagal stimulation and propranolol.

The left anterior descending coronary artery was ligated in 58 open-chest anaesthetised dogs; 23 were controls, 15 were given intravenous propranolol 1 mg . kg-1 before and at 6 h intervals after coronary ligation, nine had bilateral cervical vagal nerve stimulation (VS) before and for 4 to 6 h after coronary ligation, and 11 had both VS and propranolol. None of the 20 dogs undergoing VS developed ventricular fibrillation within the first hour after coronary ligation compared to nine of the remaining 38 (P less than 0.05). Compared to controls, myocardial creatine kinase (CK) depletion in the epicardial layer of the infarct centre measured 24 h after coronary ligation was significantly less in the groups treated separately with vagal nerve stimulation and propranolol. Myocardial blood flow (MBF) measured at 15 min after coronary ligation was reduced to the normal myocardium by the interventions, but was unchanged at the infarct centre. Severely ischaemic myocardium (MBF less than or equal to 20% of normal) was better protected by the interventions than was moderately ischaemic myocardium. At 15 min after coronary ligation, the heart rate--blood pressure product (RPP) was reduced compared with controls by propranolol (18% reduction, P less than 0.05), reduced more by vagal stimulation (by 37%, P less than 0.001) and still more by vagal stimulation with propranolol (by 43%, P less than 0.001). Preservation of CK in myocardium with MBF less than or equal to 20% of normal was improved by VS and propranolol given separately roughly in proportion to reduction in RPP, but further reduction in RPP by VS and propranolol together did not improve CK levels further. We conclude that there may be an optimum level of indices of oxygen demand for preservation of very ischaemic myocardium in experimental infarction.

Case report of a potential interaction between clonidine and electroconvulsive therapy.

In a depressed man who was receiving clonidine, several ECT sessions failed to produce seizures. The author reviews clonidine's effect on noradrenergic activity, which mediates seizures induced by a wide variety of stimuli.

Physician caseloads at public mental hospitals.

OBJECTIVE: The purpose of the study was to gather baseline data on physician caseloads, particularly psychiatric caseloads, at public mental hospitals. METHOD: A 26-item survey was sent to medical directors of public mental hospitals in the United States and its territories. Questions focused on hospital demographics, programs, and medical staffing. Survey data were analyzed and descriptive statistics were determined. RESULTS: Of 295 surveys mailed, 195 (66%) usable surveys were returned. The hospitals had a mean bed capacity of 347 (SD=301, range=10-1,926), a mean current patient population of 321 (SD=277, range=7-1,815), and 950 mean annual admissions (SD=891, range=3-5,100). Acute care was the most common treatment program (81%), followed by long-term care (71%). A wide range of psychiatric caseloads by type of program existed, with approximately equal mean and median amounts. CONCLUSIONS: The caseloads reported were, overall, reasonable and expected by the type of treatment program. The range of caseloads, however, included extremely high outliers that raise concerns about the quality of care delivered.

Synthesis and biological evaluation of phosphonamidate peptide inhibitors of enkephalinase and angiotensin-converting enzyme.

The effectiveness of phosphonamidate peptide analogues as inhibitors of rat kidney or human brain metalloendopeptidase (enkephalinase, E.C. 3.4.24.11) and angiotensin-converting enzyme (ACE, 3.4.14.1) has been explored with a series of enkephalin analogues in which the scissile Gly3-Phe4 amide bond has been replaced with a phosphonamidate moiety. These compounds exhibited good inhibitory potency against enkephalinase with several of the analogues having Ki values in the submicromolar range as contrasted to micromolar or higher toward ACE. Within a series of [(N-acylamino)methyl] phosphonamidates there was a dramatic decrease in inhibitory activity against enkephalinase as the N-acyl moiety was substituted with larger, more hydrophobic acyl groups. Likewise, the inhibitory activity of the [(N-acylamino)methyl] phosphonamidates against ACE was attenuated by larger phenylalkyl acyl functionalities, although not to the same degree as against enkephalinase. However, phosphonamidate pentapeptide analogues of (Leu)enkephalin and (D-Ala2,D-Leu5)enkephalin showed good inhibitory potency against both enzymes. Interestingly, these two (Leu)enkephalin phosphonamidate analogues were completely inactive in the electrically stimulated guinea pig ileum and mouse vas deferens preparations. Conformational factors that may be involved in this inactivity are discussed.

Synthesis and anthelmintic activity of a series of pyrazino[2,1-a][2]benzazepine derivatives.

A series of 1,2,3,4,6,7,8,12b-octahydropyrazino[2,1-alpha][2] benzazepine derivatives was prepared and the cestocidal activity of the compounds evaluated in an in vitro Taenia crassiceps screen. Many of these derivatives proved to be highly active, and 2-(cyclohexylcarbonyl)-4-oxo-1,2,3,4,6,7,8,12b- octahydropyrazino[2,1-alpha][2]benzazepine, epsiprantel (BAN) (22), was selected for further development. The structure-activity relationships are discussed.

Novel Asp32-replacement tetrapeptide analogues as potent and selective CCK-A agonists.

A series of novel CCK tetrapeptide analogues of the general formula Boc-Trp-Lys(Tac)-N(R)-(CH2)nCON(R')Phe-NH2 (Tac = o-tolylaminocarbonyl), where R,R' = H or Me and n = 1-5, have been synthesized and tested. These analogues, which lack an acidic residue at the penultimate position, demonstrated surprisingly high CCK-A receptor affinity and selectivity. The effect of N-methylation pattern on CCK-A receptor affinity showed consistent trends for analogues in which n = 1, 2, or 3, with the di-N-methylated analogues having the highest affinity in each case. However, none of these analogues had full agonist activity, as measured by percent maximal PI hydrolysis. Two conformationally constrained analogues also demonstrated high CCK-A receptor affinity and selectivity, as well as nearly maximal agonist activity. In addition, one of these conformationally-constrained analogues demonstrated anorectic activity in rats.

Tetrapeptide CCK agonists: structure-activity studies on modifications at the N-terminus.

We had reported earlier on a novel series of potent and selective tetrapeptide cholecystokinin-A (CCK-A) agonists of the general structure Boc-Trp-Lys[epsilon-Y]-Asp-N(R)PheNH2 [Y = amides, ureas; R = H, Me] that were potent anorectic agents in rats. In an effort to optimize the potency, selectivity, stability, and efficacy of our lead candidate A-71623 [R = Me, Y = o-tolylaminocarbonyl; Tac] toward development of a clinical candidate, we have explored a series of analogues in which the N-terminal Boc functionality was systematically replaced with various amides, ureas, carbamates, and sulfonamides of differing size, hydrophobicity, and stereoelectronic properties. In general, these analogues maintained good potency and selectivity for the CCK-A receptor (guinea pig pancreas), as well as potent anorectic activity in rats. Those analogues exhibiting equal or superior activity compared to A-71623 but differing physicochemical properties may represent superior drug candidates.

Ligands for brain cholinergic channel receptors: synthesis and in vitro characterization of novel isoxazoles and isothiazoles as bioisosteric replacements for the pyridine ring in nicotine.

Ligands which activate neuronal nicotinic acetylcholine receptors (nAChRs) represent a potential approach for the palliative treatment for the symptoms of memory loss associated with Alzheimer's disease (AD). Based upon this approach, a series of novel 3,5-disubstituted isoxazoles and isothiazoles were prepared and evaluated in vitro as cholinergic channel activators (ChCAs) of neuronal nAChRs. Many of the 3-substituted 5-(2-pyrrolidinyl)isoxazoles were found to have nanomolar binding affinities comparable to (S)-nicotine (2a) in a preparation of whole rat brain. However, in a paradigm measuring the evoked release of [3H]dopamine from a preparation of rat striatum, there were differences in the agonist potencies and efficacies of these analogues relative to 2a. The differences in agonist potency observed between compounds of comparable binding potency may be due to differences in ligand interactions with various subtypes of neuronal nAChRs.

Anhydrolide macrolides. 2. Synthesis and antibacterial activity of 2,3-anhydro-6-O-methyl 11,12-carbazate erythromycin A analogues.

A series of 3-descladinosyl-2,3-anhydro-6-O-methylerythromycin A 11, 12-cyclic carbazate analogues was prepared and evaluated for antibacterial activity. These 2,3-anhydro macrolides were found to be potent antibacterial agents in vitro against macrolide-susceptible organisms including Staphylococcus aureus 6538P, Streptococcus pyogenes EES61, and Streptococcuspneumoniae ATCC6303. These compounds were also very active against some organisms that show macrolide resistance (S. aureus A5177, S. pyogenes PIU2584, and S. pneumoniae 5649). The compounds generally showed poor activity against organisms with constitutive MLS resistance. Selected compounds were evaluated in vivo in mouse protection studies. Although most of the compounds tested in vivo showed poor efficacy, two compounds, 38 and 57, were more active than clarithromycin against S. pneumoniae ATCC6303.

Anhydrolide macrolides. 1. Synthesis and antibacterial activity of 2,3-anhydro-6-O-methyl 11,12-carbamate erythromycin A analogues.

A series of 3-descladinosyl-2,3-anhydro-6-O-methylerythromycin A 11, 12-carbamate analogues have been synthesized and evaluated for antibacterial activity. These compounds were found to be potent antibacterial agents against Gram-positive organisms in vitro, many having MIC values below 1 microg/mL for the macrolide-susceptible Staphylococcus aureus, Streptococcus pyogenes, and Streptococcus pneumoniae, as well as improved activity compared to erythromycin A against the inducibly MLS (macrolide, lincosamide, and streptogramin B)-resistant organisms. Structure-activity studies revealed that arylalkyl carbamates with two and four carbon atoms between the aromatic moiety and carbamate nitrogen have the best in vitro activity. All of the C-10 epi analogues evaluated were found to have substantially less activity than the corresponding natural C-10 isomer. Several analogues demonstrated moderate antibacterial activity against the constitutively resistant S.aureus A-5278, S. pneumoniae5979, and S.pyogenes 930. However, despite potent in vitro activity, these analogues showed only moderate in vivo activity in mouse protection studies.

Breast thermography is a noninvasive prognostic procedure that predicts tumor growth rate in breast cancer patients.

Our recent retrospective analysis of the clinical records of patients who had breast thermography demonstrated that an abnormal thermogram was associated with an increased risk of breast cancer and a poorer prognosis for the breast cancer patient. This study included 100 normal patients, 100 living cancer patients, and 126 deceased cancer patients. Abnormal thermograms included asymmetric focal hot spots, areolar and periareolar heat, diffuse global heat, vessel discrepancy, or thermographic edge sign. Incidence and prognosis were directly related to thermographic results: only 28% of the noncancer patients had an abnormal thermogram, compared to 65% of living cancer patients and 88% of deceased cancer patients. Further studies were undertaken to determine if thermography is an independent prognostic indicator. Comparison to the components of the TNM classification system showed that only clinical size was significantly larger (p = 0.006) in patients with abnormal thermograms. Age, menopausal status, and location of tumor (left or right breast) were not related to thermographic results. Progesterone and estrogen receptor status was determined by both the cytosol-DCC and immunocytochemical methods, and neither receptor status showed any clear relationship to the thermographic results. Prognostic indicators that are known to be related to tumor growth rate were then compared to thermographic results. The concentration of ferritin in the tumor was significantly higher (p = 0.021) in tumors from patients with abnormal thermograms (1512 +/- 2027, n = 50) compared to tumors from patients with normal thermograms (762 +/- 620, n = 21). Both the proportion of cells in DNA synthesis (S-phase) and proliferating (S-phase plus G2M-phase, proliferative index) were significantly higher in patients with abnormal thermograms. The expression of the proliferation-associated tumor antigen Ki-67 was also associated with an abnormal thermogram. The strong relationships of thermographic results with these three growth rate-related prognostic indicators suggest that breast cancer patients with abnormal thermograms have faster-growing tumors that are more likely to have metastasized and to recur with a shorter disease-free interval.

Breast carcinoma and the role of iron metabolism. A cytochemical, tissue culture, and ultrastructural study.

Transferrin receptors on proliferating and malignant cells are well documented. Iron is an essential micronutrient for cell growth that plays an important role in energy metabolism and DNA synthesis. Malignant cells requiring more iron modulate a transferrin receptor. Iron-bound transferrin interacts with this receptor, facilitating the transport of iron across the cell membrane. Transferrin is a glycoprotein and is the chief iron transport protein in mammalian blood. The more aggressive the tumor, the higher the transferrin receptor levels and the greater the proliferative index. We have found by cytochemical and ultrastructural studies that ferritin, an iron storage protein, is increased in breast cancer tissue. Anaplastic tumors have higher tissue ferritin levels. Tissue ferritin concentration may be an indirect method of measuring transferrin receptors and thus might be an index of proliferation and a prognostic indicator. Transferrin may be used as a carrier to target toxic therapy selectively to tumor tissue. A platinum transferrin complex (MPTC-63) has been developed and shown to be cytostatic in tissue culture, animal, and human studies. It also sensitizes tissue to agents that produce free radicals, such as adriamycin, and thus is synergistic with other drugs and radiation. Other transferrin complexes and conjugates of gallium, indium, and daunorubicin have also shown growth inhibition in tissue culture and animals. Human studies are in progress. By studying iron metabolism in breast cancer, we may be able to selectively inhibit tumor growth without toxic effects, and with other tumor biologic data be better able to select the stage I patient for adjuvant therapy.

A novel cell culture technique for electron microscopy.

A simplified technique for the monolayer growth of cultured cells and their in situ embedment on the inner surface of the pyramidal portion of the Beem capsule for electron microscopy has been developed. The results demonstrated that the cell monolayers grew well on the surface of the Beem capsule and could be embedded in situ. Electron micrographs showed cells in their natural state of contact with one another. The plasma membrane and intracellular organelles were well preserved. This method minimizes many difficult steps and eliminates the disruption of cells by scraping, pelleting, or enzymatic reaction to remove them.

Antineoplastic drugs that interfere with iron metabolism in cancer cells.

Normal iron metabolism can be perturbed with iron chelators, toxic metals that bind to transferrin, toxic metals bound to transferrin or antineoplastic agents covalently linked to transferrin. These agents cause significant inhibition of tumor cell growth in cell culture and have been shown to have significant in vivo antineoplastic activity. Cell culture studies showed that deferoxamine mesylate inhibits cell growth and division in both the MCF-7 human breast and HeLa human cervical carcinoma cell lines. Animal studies demonstrated that when deferoxamine mesylate is injected intravenously into rats that are on a low iron diet, there is a significant reduction in the growth of 13762NF mammary adenocarcinomas. Gallium, indium and the antineoplastic agent cisplatin were bound to the iron binding site of transferrin and inhibit the growth of malignant carcinoma cell lines. Gallium-transferrin and indium-transferrin were at least 10 times more inhibitory to both MCF-7 and HeLa cell lines than their free salts. Further cell culture studies demonstrated that cisplatin-transferrin complexes act synergistically with doxorubicin to inhibit the growth of cultured MCF-7 cells. In a Phase I clinical trial of cisplatin-transferrin complex there was a 36% (four of 11 patients) response rate in breast cancer patients with advanced disease. In a second clinical study the sequential administration of deferoxamine mesylate (2 days at 6 g/day in 8 hrs), cisplatin-transferrin complex (7 days at 500 mg/day) and FAC (5-fluorouracil, doxorubicin and cyclophosphamide at 450, 45 and 450 mg/m2, respectively) to advanced breast cancer patients resulted in partial responses in seven of eight patients treated. Future work will concentrate on substituting transferrin based agents with daunorubicin or doxorubicin attached to the surface of the transferrin, and gallium or indium bound to the iron binding site, to increase efficacy of the second component of the sequential combination chemotherapy.

The effect of anterior transposition of the inferior oblique muscle.

The effect of anterior transposition of the insertion of the inferior oblique muscle was compared with the results from conventional inferior oblique muscle recession in 50 patients. Even though both groups of patients had a similar degree of overaction preoperatively, postoperative inferior oblique muscle action was weaker (P < .01) and upgaze more limited P < .01) in the anterior transposition group. These data suggest that anterior transposition serves to convert the inferior oblique muscle from an elevator to a depressor on attempted elevation. Because anterior transposition is such a powerful weakening operation, we suggest that it be reserved for patients with moderate to severe inferior oblique muscle overaction. To avoid postoperative hypotropia in upgaze, anterior transposition should be performed in both eyes for bilateral inferior oblique muscle overaction and not unilaterally.

Quality in residency training: toward a broader, multidimensional definition.

The authors propose a general definition of quality in residency train ing and describe a process for developing the definition. The definition and process go beyond the usual concerns with educational structures and processes and are customer-centered, outcomes-oriented, and consistent with views of quality widely applied in health care, manufacturing, and other businesses. The process begins by listing some of the more important customers of a training program--trainees, patients, funding agencies, health care employers, medical schools and teaching hospitals, and professional organizations--and then considers the needs of those customers. The result is a definition of quality in residency training that is multidimensional and includes aspects of residency training related to the clinical proficiency of trainees, socioeconomic dimensions of training, and ethical concerns. While some measures exist to put this multidimensional definition of quality into operation, much work will be needed to make it useful for the training of residents.

Inhibitory effect of deferoxamine mesylate and low iron diet on the 13762NF rat mammary adenocarcinoma.

The iron chelator deferoxamine mesylate has been shown to inhibit the growth of a variety of human malignant cell lines and the rat 13762NF mammary adenocarcinoma cell line. In vivo studies in mice have also demonstrated that an iron deficiency induced by either feeding a low iron diet or injecting the iron chelator deferoxamine mesylate decreases tumor growth. In this study Fisher rats were transplanted with the 13762NF mammary adenocarcinoma and divided into four groups: normal diet, normal diet plus deferoxamine mesylate treatment, low iron diet and low iron diet plus deferoxamine mesylate treatment. The measurements of tumor size and body weight were recorded weekly. We found that treatment with either deferoxamine mesylate or a low iron diet decreased rat tumor growth, but the greatest inhibitory effect on tumor growth occurred when the rats were treated with deferoxamine mesylate injections plus fed a low iron diet. These treatments did not significantly inhibit the weight gain of the rats. At the end of the experiments measurement of serum iron proved that these treatments caused iron deficiency, but there was no significant treatment related alteration in blood hematocrit. We therefore concluded that deferoxamine mesylate may be a useful chemotherapeutic agent in the treatment of breast cancer, when used in combination with standard chemotherapeutic regiments or with other agents that interfere with iron metabolism, and further that the restricting of iron intake should be considered when planning chemotherapy for all cancer patients.

Expression of transferrin receptor and ferritin H-chain mRNA are associated with clinical and histopathological prognostic indicators in breast cancer.

It is well known that iron plays an essential role in many biochemical reactions and that rapidly growing cells require more iron for their growth and metabolism than resting cells. Transferrin and its receptor are required for entry of iron into the cell. In contrast, ferritin is a cellular storage protein whose main function is to sequester excess ferric iron and thus prevent high concentrations of soluble ferric iron from becoming toxic to the cell. However, the clinical significance of both transferrin receptor and ferritin mRNA levels have not previously been described in tumors from breast cancer patients. In this study, tumor tissue mRNA levels of transferrin receptor and ferritin were quantitated on forty-two breast cancer patients. A highly sensitive non-radioisotopic cDNA polymerase chain reaction assay was used to quantitate expression of mRNA. The expression of glyceraldehyde-3-phosphate dehydrogenase served as the control. In the tumor tissue from the 42 breast cancer patients the transferrin receptor mRNA levels were significantly correlated to the ferritin H-chain mRNA levels (Spearman correlation r = 0.5433, p = 0.0002; Pearson correlation r = 0.6276, p < 0.0001). The level of amplified transferrin receptor complementary DNA was related to differentiation (ANOVA, p = 0.042) with poorly differentiated tumors having high levels of transferrin receptor mRNA. Further, the levels of amplified gene for ferritin heavy chain complementary DNA was directly related to axillary lymph nodes status (Student's t-test, p = 0.044), presence of metastatic disease (Student's t-test, p = 0.046) and clinical stage (stage I + stage II versus stage III + stage IV; Student's t-test, p = 0.0181). These results demonstrate that non-radioisotopic RT-PCR is a very sensitive method for determining mRNA levels in tumor tissue. Additionally, the quantitation of expression of transferrin receptor and ferritin heavy chain mRNA may be useful for assessing prognosis and guiding therapeutic decisions in breast cancer patients.

Doxorubicin-gallium-transferrin conjugate overcomes multidrug resistance: evidence for drug accumulation in the nucleus of drug resistant MCF-7/ADR cells.

Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin (ADR) uptake as a result of increased efflux, increased intracellular sequestration, and decreased membrane permeability. In this study, we investigated whether conjugation of ADR to transferrin (Tf) could overcome MDR in breast cancer cells. The multidrug resistant MCF-7/ADR breast cancer cell line was over 1000-fold more resistant to ADR, than its parental MCF-7 cell line, as determined by 3[H]-thymidine assay. The MCF-7/ADR cell line also expressed both MDR1 and MRP genes, as detected by RT-PCR. The ADR was coupled using a glutaraldehyde technique to human transferrin saturated with either ferric chloride (Fe-Tf) or gallium nitrate (Ga-Tf). These conjugates were tested for cytotoxicity on both MCF-7 and MCF-7/ADR cells after 6 days of incubation. The doxorubicin-gallium-transferrin conjugate (ADR-Ga-Tf) exhibited approximately the same inhibitory effect as ADR on MCF-7 cells with IC50s of 2.34 x 10(-3) microM and 1.42 x 10(-3) microM, respectively. However in MCF-7/ADR cells ADR-Ga-Tf reversed resistance to free ADR and decreased 100-fold the IC50 from 8.98 microM with free ADR to 9.52 x 10(-2) microM. ADR-Fe-Tf was 10-fold more inhibitory to MCF-7/ADR cells than free ADR. Compared to Ga-Tf, ADR-Ga-Tf was 500- and 3000-fold more inhibitory to MCF-7 and MCF-7/ADR cells, respectively. These results demonstrated that ADR-Ga-Tf reverses resistance to free ADR and Ga-Tf in MCF-7/ADR cells. The distribution of ADR in both cell lines was examined by fluorescence microscopy. It was noted that ADR mainly accumulated in the cytoplasm around the nucleus in MCF-7/ADR cells, but in both the cytoplasm and nucleus of MCF-7 cells. However the conjugate of ADR-Ga-Tf allowed ADR to accumulate in the cytoplasm and nucleus of both the MCF-7/ADR and MCF-7 cells. Further investigation of MDR1 and MRP genes expression by RT-PCR demonstrated that Ga-Tf decreased expression of the MRP more than the MDR1 gene. Therefore the reversal of resistance to ADR by the ADR-Ga-Tf conjugate is mediated by the transferrin receptor transmembrane transport mechanism, redistribution of ADR into the nucleus of ADR resistant MCF-7/ADR cells and inhibition of MRP gene expression.