Ultrastructural changes induced in Walker carcinosarcoma by treatment with dihematoporphyrin ester and light in animals with diabetes mellitus

The purpose of this study was to evaluate by electron microscopy the tumoral fine structure changes induced by photodynamic therapy (PDT) in diabetic animals. Walker -256 carcinosarcoma harvested from animals with/without diabetes mellitus exposed to PDT (Photofrin II/5 mg/kg and 24 hrs later He-Ne laser irradiation/632.8 nm; 10 mW) and examined by electron microscopy showed different degrees of lesions in the nucleus and cytoplasmic fine structure. The ultrastructural changes induced by PDT in animals with diabetes mellitus bearing carcinosarcoma are characterized by: lysis of chromatin situated on the central zone of nucleus; swelling and vacuolization of mitochondria; formation of phagosome--like structures; myelin figures; degenerescence and disappearance of cytoplasmic organelles. Summing up, the data presented in this work demonstrate that the exposure to three doses of PDT produces changes in tumoral fine structure, increases survival rate and reduces incidence of carcinosarcoma in rats with diabetes mellitus.     

Subcellular localization of Photofrin determines the death phenotype of human epidermoid carcinoma A431 cells triggered by photodynamic therapy: when plasma membranes are the main targets

Photodynamic therapy (PDT) is a kind of photochemo-therapeutic treatment that exerts its effect mainly through the induction of cell death. Distinct types of cell death may be elicited by different PDT regimes. In this study, the mechanisms involved in the death of human epidermoid carcinoma A431 cells triggered by PDT with Photofrin (a clinically approved photosensitizer) were characterized. Photofrin distributes dynamically in A431 cells; the plasma membranes and Golgi complex are the main target sites of Photofrin after a brief (3 h) and prolonged (24 h) incubation, respectively. Cells with differentially localized Photofrin displayed distinct death phenotypes in response to PDT. The effects of PDT on cells with plasma membrane-localized Photofrin were further studied in details. Cells stopped proliferating post PDT at Photofrin dose >7 micro g/ml, and at higher dose (28 micro g/ml) plasma membrane disruption and cell swelling were observed immediately after PDT. Dramatic alterations of several important signaling events were detected in A431 cells post Photofrin-PDT, including (i) immediate formation of reactive oxygen species (ROS), (ii) rapid activation of c-Jun N-terminal kinase, (iii) delayed activation of caspase-3 and cleavage of polyADP-ribose polymerase and p21-activated kinase 2, and (iv) loss of mitochondrial membrane potential. Intriguingly, the characteristics of typical apoptosis such as phosphatidylserine externalization and DNA fragmentation were not detected in the cell death process caused by this PDT regime. In conclusion, our results show that when plasma membranes are the main targets, Photofrin-PDT can lead to instant ROS formation and subsequent activation of downstream signaling events similar to those elicited by many apoptotic stimuli, but the damage of plasma membranes renders the death phenotype more necrosis like.     

The effect of laser activation of 5,10,15,20-tetra-sulphophenyl-porphyrin loaded in K562 cells and human normal mononuclear cells

Photodynamic therapy (PDT) is a relatively new type of treatment in cancer, based on a photosensitizer, visible light and molecular oxygen. Reactive oxygen species are generated, causing tumor cells death by apoptosis or necrosis. Significant nowadays research efforts are focused on finding new photosensitizers with antineoplastic activity and an acceptable toxicological profile. Although consistent information exists regarding PDT in solid tumors, relatively few data are available for PDT of blood cancers. Therefore, we carried out a comparative study on lymphoblastic K562 cells and human normal peripheral blood mononuclear cells (PBMC) treated at a density of 2 x 10(5) cells/mL with 5,10,15,20-tetra-sulphophenyl-porphyrin (TSPP) and then irradiated with He-Ne laser light (lamda = 632.8 nm). The following cell functions were investigated: viability, multiplication, RNA synthesis, total RNA levels and apoptosis. After irradiation, the viability of TSPP-loaded tumor cells decrease, the multiplication rate and the total RNA level are drastically reduced and cells undergo apoptosis. TSPP alone loaded into cells but not activated by irradiation, does not affect these cell parameters. Human normal PBMC subjected to TSPP loading and laser-irradiation develop a different cellular response, their viability and proliferative capacity not being altered by experimental PDT. Accordingly, it appears that TSPP is a non-aggressive compound for cellular physiology and becomes cytotoxic only by irradiation; moreover laser-activated TSPP affects only cells that have a tumoral pattern.     

The effect of chemotherapy on the in vivo frequency of glycophorin A ‘null’ variant erythrocytes

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin     

Effects of probucol on lipid metabolism and secretion in long-term cultures of adult rat hepatocytes

To study the effects of probucol on hepatic lipid metabolism, we used adult rat hepatocytes cultured on a feeder layer of 3T3 cells lethally treated with mitomycin C. These cultures synthesize and secrete for at least 2 weeks various lipids from [14C]acetate and [14C]oleate precursors. Treatment with 20 micrograms/ml of probucol for 7 and 14 days decreased the secretion of various radiolabeled lipid species to the culture medium and produced an intracytoplasmic accumulation of triacylglycerol droplets. The lipids whose secretion was most decreased were free and esterified cholesterol (50-70% reduction). Secretion of triacylglycerols and phospholipids was also reduced but to a lower extent. Intracytoplasmic triacylglycerols accumulated and the activity of glycerol phosphate dehydrogenase, a marker enzyme of glycerolipid synthesis, also increased (35-56%). The total incorporation of both radioactive precursors into free and esterified cholesterol and phospholipids was reduced 20-60%. Our data show that 2-week treatment of 3T3-hepatocyte cultures with pharmacological concentrations of probucol reduces significantly lipid secretion and suggest that at least part of the in vivo hypolipidemic effect of probucol could be attributed to a decrease in the secretion of lipids (i.e., lipoproteins) by hepatocytes.     

The effect of chemotherapy on the in vivo frequency of glycophorin A 'null' variant erythrocytes

A human in vivo somatic cell assay based on the enumeration of variant erythrocytes lacking expression of an allelic form of the cell-surface sialoglycoprotein, glycophorin A, was applied to the study of blood samples from patients obtained prior to, during, and following chemotherapy for malignant disease in order to determine the effect of mutagenic chemical agents on the frequency of variant cells. In 22 patients assayed prior to therapy, the mean variant cell frequency was 11.9 per million, which was not significantly different from that observed in healthy controls. In an initial cross-sectional survey, blood samples were obtained at various times during and after therapy from 30 patients diagnosed with a variety of malignancies who were treated with one or more known mutagenic agents including adriamycin, bleomycin, cis-platinum, cyclophosphamide, dacarbazine, etoposide, lomustine, mechlorethamine, melphalan, mitomycin C, and procarbazine. Significant elevations in the mean frequency of variant cells over pre-therapy and normal levels were observed in samples obtained during and after therapy. In a time-series study, 14 breast cancer patients treated with CAF (cyclophosphamide, adriamycin, 5-fluorouracil), CMF (cyclophosphamide, methotrexate, 5-fluorouracil), or VMF (vinblastine, methotrexate, 5-fluorouracil) adjuvant chemotherapy were sampled repeatedly during and after therapy. For the CAF and CMF patients an increase in the frequency of variant cells was observed with a lag in the appearance of induced variants after initiation of therapy; variant frequencies gradually increased during therapy reaching a maximum at or shortly after the end of therapy, then declined to near pre-therapy levels within 6 months. The maximum level of induced variants ranged from 2- to 7-fold over pre-therapy or normal levels depending on the combination of agents used. The breast cancer patients treated with both adriamycin and cyclophosphamide showed consistent elevations in the frequency of variant cells; patients treated only with cyclophosphamide showed lower and more variable elevations. The data demonstrate that mutagenic chemotherapy agents induce elevated levels of glycophorin A variant erythrocytes consistent with the hypothesis that variant cells result from somatic mutation. The elevations in variant cells were transient, suggesting that these agents primarily affect the rapidly cycling committed erythroid cell population.     

Photodynamic effects of porphyrin and chlorin photosensitizers in human colon adenocarcinoma cells

Photodynamic therapy (PDT) is a cancer treatment involving systemic administration of a tumor-localizing photosensitizer; this, when activated by the appropriate wavelength of light, interacts with molecular oxygen to form a toxic, short-lived species known as singlet oxygen, which is thought to mediate cellular death. Photofrin, a complex mixture of porphyrin oligomers has recently received FDA approval for the photodynamic treatment of esophageal and endobronchial carcinoma, but its photodynamic and toxicity profiles are far from ideal. In the present study we evaluated a series of porphyrin-based PSs, some of which newly synthesized by our group, with the aim to identify agents with more favorable characteristics. For the most effective compounds in the porphyrin series, chlorin analogs were also synthesized; for comparison, the screening also included Photofrin. Cytotoxicity studies were performed by the MTT assay on a cultured human colon adenocarcinoma cell line (HCT116); the results indicate that the 3,4,5-trimethoxyphenyl, 3OH- and 4OH-phenyl, and the sulfonamidophenyl derivatives are significantly more potent than Photofrin. Flow cytometric studies and fluorescence microscopy indicate that in PDT-treated HCT116 cells death occurs mainly by apoptosis. In summary, novel PSs described in the present study, belonging both to the porphyrin and chlorin series, have proven more effective than Photofrin in killing colon cancer cells in vitro; extending these observation to in vivo models, particularly regarding the deeper reaching chlorin derivatives, might lead to significant advances in the development of tumor PDT.     

Characterization of photodynamic therapy responses elicited in A431 cells containing intracellular organelle‐localized photofrin

Photodynamic therapy (PDT), a photochemotherapeutic regimen used to treat several diseases, including cancer, exerts its effects mainly through induction of cell death. Using human epidermoid carcinoma A431 cells as a model, we previously showed that distinct cell death types could be triggered by protocols that selectively delivered Photofrin (a clinically approved photosensitizer) to different subcellular sites (Hsieh et al. [2003] J Cell Physiol 194: 363–375]. Here, the responses elicited by PDT in A431 cells containing intracellular organelle‐localized Photofrin were further characterized. Two prominent cell phenotypes were observed under these conditions: one characterized by perinuclear vacuole (PV) formation 2–8 h after PDT followed by cell recovery or shrinkage within 48 h, and a second characterized by typical apoptotic features appearing within 4 h after PDT. DCFDA‐sensitive reactive oxygen species formed proximal to PVs during the response to PDT, covering areas in which both endoplasmic reticulum (ER) and the Golgi complex were located. Biochemical analyses showed that Photofrin‐PDT also induced JNK activation and altered the protein secretion profile. A more detailed examination of PV formation revealed that PVs were derived from the ER. The alteration of ER structure induced by PDT was similar to that triggered by thapsigargin, an ER Ca²⁺‐ATPase inhibitor that perturbs Ca²⁺ homeostasis, suggesting a role for Ca²⁺ in the formation of PVs. Microtubule dynamics did not significantly affect PV formation. This study demonstrates that cells in which intracellular organelles are selectively loaded with Photofrin mount a novel response to ER stress induced by PDT. J. Cell. Biochem. 111: 821–833, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of selenite combined with chemotherapeutic agents on the proliferation of human carcinoma cell lines

Selenite is frequently used in combination with cancer chemotherapeutic agents to reduce side effects. However, the cytoprotective activity of selenite may also reduce the efficacy of chemotherapeutic drugs on tumor cells. This study was designed to examine the effects of selenite combined with cytotoxic agents used in clinical protocols [e.g., doxorubicine, docetaxel, 5-fluorouracil (5-FU), methotrexate (MTX), mafosphamide, mitomycin C, gemcitabine, etoposide, cisplatin, irinotecan, and oxaliplatin] on the proliferation of various carcinoma cell types. The data demonstrated that selenite had no marked effects on the antiproliferative activity of docetaxel, doxorubicine, 5-FU, MTX, and mafosphamide in MDA-MB-231 breast cancer cells. Likewise, no consistent changes were observed in A549 lung cancer cell proliferation when selenite was combined with cisplatin, etoposide, gemcitabine, or mitomycin C. On the other hand, selenite potentiated the cytotoxicity of 5-FU, oxaliplatin, and irinotecan in HCT116 colon cancer cells by approx 1.1-fold, 2.7-fold, and 2.6-fold, respectively. In SW620 colon cancer cells, selenite induced a 1.5-fold and 4.3-fold increase of the antiproliferative activity of 5-FU and oxaliplatin, respectively. Whereas irinotecan showed no effects on SW620 cell growth, a combination with selenite resulted in 23% inhibition. Our results indicate that selenite did not reduce the antiproliferative activity of chemotherapeutic agents in vitro. In addition, selenite was able to increase the inhibitory activity of docetaxel in A549 lung cancer cells, and of 5-FU, oxaliplatin, and irinotecan in HCT116 and SW620 colon cancer cells implying selenite is potentially useful as an adjuvant chemotherapeutic agent.     

Bax is essential for Drp1‐mediated mitochondrial fission but not for mitochondrial outer membrane permeabilization caused by photodynamic therapy

Bcl‐2 family proteins are critical for the regulation of apoptosis, with the pro‐apoptotic members Bax essential for the release of cytochrome c from mitochondria in many instances. However, we found that Bax was activated after mitochondrial depolarization and the completion of cytochrome c release induced by photodynamic therapy (PDT) with the photosensitizer Photofrin in human lung adenocarcinoma cells (ASTC‐a‐1). Besides, knockdown of Bax expression by gene silencing had no effect on mitochondrial depolarization and cytochrome c release, indicating that Bax makes no contribution to mitochondrial outer membrane permeabilization (MOMP) following PDT. Further study revealed that Bax knockdown only slowed down the speed of cell death induced by PDT, indicating that Bax is not essential for PDT‐induced apoptosis. The fact that Bax knockdown totally inhibited the mitochondrial accumulation of dynamin‐related protein (Drp1) and Drp1 knockdown attenuated cell apoptosis suggest that Bax can promote PDT‐induced apoptosis through promoting Drp1 activation. Besides, Drp1 knockdown also failed to inhibit PDT‐induced cell death finally, indicating that Bax‐mediated Drp1's mitochondrial translocation is not essential for PDT‐induced cell apoptosis. On the other hand, we found that protein kinase Cδ (PKCδ), Bim L and glycogen synthase kinase 3β (GSK3β) were activated upon PDT treatment and might contribute to the activation of Bax under the condition. Taken together, Bax activation is not essential for MOMP but essential for Drp1‐mediated mitochondrial fission during the apoptosis caused by Photofrin‐PDT. J. Cell. Physiol. 226: 530–541, 2011. © 2010 Wiley‐Liss, Inc.     

Photodynamic effect of deuteroporphyrin IX and hematoporphyrin derivatives on single neuron

The photodynamic effects of 6 new deuteroporphyrin IX derivatives with different amphiphilicity and lipophilicity, as well as effects of known hematoporphyrin derivatives Photofrin II and Photoheme on isolated crayfish mechanoreceptor neurons were studied. After 30 min photosensitization, neurons were irradiated with He-Ne laser (632.8 nm, 0.3 W/cm(2)), and changes in their firing frequency were recorded. Neuron firing was shown to be very sensitive to photodynamic effect of the studied deuteroporphyrin IX derivatives causing irreversible firing abolition at pikomolar concentrations while Photoheme and Photofrin II were effective in the nanomolar range. The most effective sensitizers were 4-(1-methyl-3-hydroxybutyl)- and 4-(1-methyl-2-acetyl-3-oxobutyl)-deuteroporphyrins. Extinction and amphiphilicity were shown to be the most important properties determining photodynamic efficiency of the studied photosensitizers.     

New porphyrin amino acid conjugates: Synthesis and photodynamic effect in human epithelial cells

The efficacy of new porphyrin amino acid conjugates as photosensitizers for photodynamic therapy (PDT) were assayed in vitro on tumoral (HeLa) and on non tumoral (HaCaT) human cell lines. The conjugates stable in liposomes are able to penetrate efficiently in the cytoplasm of cultured cancer and normal cells. No dark cytotoxicity is observed at the same concentration used for PDT cell treatment and during long incubation time (24 h). The cell survival after the PDT treatment with visible light is dependent upon light exposure level and compound concentration. The tested compounds show higher photocytotoxicity in tumoral HeLa cells than in no tumoral HaCaT cells. The results suggest that these amino acid porphyrin conjugates are potential photosensitizers for PDT.     

Role of mitochondria in cell death induced by Photofrin-PDT and ursodeoxycholic acid by means of SLIM.

The present study was undertaken to find new ways to improve efficacy of photodynamic therapy (PDT). We investigated the combinatory effect of the photosensitizer Photofrin and ursodeoxycholic acid (UDCA). UDCA is a relatively non-toxic bile acid which is used inter alia as a treatment for cholestatic disorders and was reported to enhance PDT efficiency of two other photosensitizers. Since besides necrosis and autophagic processes apoptosis has been found to be a prominent form of cell death in response to PDT for many cells in culture, several appropriate tests, such as cytochrome c release, caspase activation and DNA fragmentation were performed. Furthermore spectral resolved fluorescence lifetime imaging (SLIM) was used to analyse the cellular composition of Photofrin and the status of the enzymes of the respiratory chain. Our experiments with two human hepatoblastoma cell lines revealed that the combination of Photofrin with UDCA significantly enhanced efficacy of PDT for both cell lines even though the underlying molecular mechanism for the mode of action of Photofrin seems to be different to some extent. In HepG2 cells cell death was clearly the consequence of mitochondrial disturbance as shown by cytochrome c release and DNA fragmentation, whereas in Huh7 cells these features were not observed. Other mechanisms seem to be more important in this case. One reason for the enhanced PDT effect when UDCA is also applied could be that UDCA destabilizes the mitochondrial membrane. This could be concluded from the fluorescence lifetime of the respiratory chain enzymes which turned out to be longer in the presence of UDCA in HepG2 cells, suggesting a perturbation of the mitochondrial membrane. The threshold at which PDT damages the mitochondrial membrane was therefore lower and correlated with the enhanced cytochrome c release observed post PDT. Thus enforced photodamage leads to a higher loss of cell viability.     

Single Cell FRET Imaging for Determination of Pathway of Tumor Cell Apoptosis Induced by Photofrin-PDT

Apoptosis is an important cellular event that plays a key role in pathogeny and therapy ofmany diseases. Apoptosis has been associated with photodynamic therapy (PDT) and itspathway is important in the mechanistic study of PDT. We show that single cell fluorescentimaging can be used to determine the pathway of PDT- induced tumor cell apoptosis. In thisstudy, ASTC-a-1 tumor cells transfected by plasmid DNA SCAT3 were treated by Photofrin-PDT.The intracellular distribution of Photofrin was observed using a confocal microscope. Theactivations of caspase-3 and caspase-8 were dynamically observed using fluorescence resonanceenergy transfer (FRET). Our experimental results show that the Photofrin molecules arelocalized in cell mitochondria, and that after PDT caspase-3 was activated rapidly whilecaspase-8 remained inactive. These results demonstrate that the tumor cell apoptosis inducedby Photofrin-PDT was directly initiated from the mitochondrial pathway.     

Intratumoural light distribution in an experimental mouse tumour irradiated by a diffuse-light irradiator compared with unilateral helium-neon light for photodynamic therapy

Investigations on dosimetry in photodynamic therapy (PDT) of experimental mouse tumours transplanted into the right hind leg revealed a significant variability in the fluence rate reaching tumour cells in different parts of the tumour when irradiated by a 50 mW collimated He-Ne laser. Based on intratumoural fibre-optical light measurements, a new irradiation source was constructed, in which variability in the radiant energy fluence rate between different parts of the solid tumour was reduced. The new diffuse-light irradiator was constructed basically from two concentric water chambers surrounded by three linear 3000 W xenon flash lamps. The outer chamber was an optical band filter, and the inner chamber contained a light-dispersing solution of lipid droplets which created an isotropic light field in which the tumours were submerged for PDT. Compared with unilateral He-Ne laser irradiation, an enhancement factor of 7.3 in radiance was obtained for the diffuse-light irradiator measured in the tumour. The new apparatus provides a nearly isotropic light field for in vivo experimental PDT.     

Increased vinblastine binding to membrane vesicles from multidrug-resistant KB cells

Human KB carcinoma cells resistant to high levels of colchicine, vinblastine, vincristine, adriamycin, and actinomycin D exhibit reduced accumulation of these structurally unrelated chemotherapeutic agents (Akiyama, S.-I., Fojo, A., Hanover, J. A., Pastan, I., and Gottesman, M. M. (1985) Somatic Cell Mol. Genet. 11, 117-126; Fojo, A., Akiyama, S.-I., Gottesman, M. M., and Pastan, I. (1985) Cancer Res. 45, 3002-3007). To examine the mechanism of reduced drug accumulation in these cells, we measured [3H]vinblastine ([3H]VBL) binding to membrane vesicles made from drug-sensitive (KB-3-1), drug-resistant (KB-C4), and revertant (KB-R1) cells. Membrane vesicles from KB-C4 cells bound up to 8-fold more [3H]VBL than vesicles from the parental KB-3-1 or revertant KB-R1 cell lines. No difference in binding of [3H]dexamethasone, to which the cells are equally sensitive, was observed. The difference in [3H]VBL binding by vesicles from resistant and sensitive cells was eliminated by the addition of 10 micrograms/ml verapamil, which is known to reverse the multidrug-resistance phenotype. Drug binding by KB-C4 vesicles was osmotically insensitive, temperature-dependent, and trypsin-sensitive. Binding of [3H]VBL by KB-C4 vesicles was inhibited by vinblastine, vincristine, and daunomycin (in decreasing order). Dexamethasone at 100 microM, colchicine at 100 microM, and actinomycin D at 100 microM did not significantly inhibit [3H]VBL accumulation. No significant differences in tubulin content were detected among vesicles from sensitive and resistant cells. These data demonstrate that membrane vesicles from multiply drug-resistant cells bind increased amounts of vinblastine.     

A rapid bioassay to determine stabilities of anticancer agents under conditions of the clonogenic assay

Summary We developed a rapid bioassay to determine in vitro drug stabilities in the clonogenic assay. The in vitro half-lives of 11 standard antitumor agents, actinomycin D, Adriamycin, bleomycin, cis-Platinum, dacarbazine, 5-fluorouracil, melphalan, mitomycin C, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), vinblastine, and vincristine, and four investigational drugs, Aziridinylbenzoquinone (AZQ) (NSC 182986), diamminecyclobutane-dicarboxylatoplatinum (CBDCA) (NSC 241240), Dihydroxyanthracenedione dihydrochloride (DHAD) (NSC 301739), and 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU) (NSC 95466), were determined under conditions of the clonogenic assay as well as under storage conditions at −40° and−196°C. BCNU and AZQ at−40°C had t1/2 of 4.7 d and 2.5 d, respectively. All other drugs were stable at −40° and −196°C with t1/2>6 wk. Under assay conditions at 37°C, actinomycin D, bleomycin, dacarbazine, 5-fluorouracil, mitomycin C, vinblastine, vincristine, and DHAD were stable, with t1/2>14 d. CBDCA, AZQ, Adriamycin, cis-Platinum, melphalan, BCNU, and PCNU had t1/2 of 94,72,29,18.5,1.8,1, and 0.5 h, respectively. This work was supported by Veterans Administration Medical Research Service and by Contract CM57710 of the National Cancer Institute.     

Modulation of P-glycoprotein phosphorylation and drug transport by sodium butyrate.

P-Glycoprotein (Pgp) expression in cell lines derived from tumors arising from cells which normally express Pgp can be increased by sodium butyrate and other differentiating agents. Although the Pgp level increased 25-fold after sodium butyrate treatment in SW620 human colon carcinoma cells, the intracellular accumulation of vinblastine, adriamycin, and actinomycin D increased rather than decreased. In contrast, colchicine showed the expected decrease in accumulation, as a result of increased efflux. Likewise, treatment of a Pgp-expressing multidrug-resistant SW620 subline with sodium butyrate resulted in active interference with Pgp function. This effect was partially reversed by phorbol esters with a resulting decrease in the accumulation of vinblastine, adriamycin, and actinomycin D. Sodium butyrate, while increasing Pgp levels, inhibited the phosphorylation of Pgp. Time course studies revealed a tight relationship between decreased Pgp phosphorylation and increased vinblastine accumulation after sodium butyrate treatment. Either treatment with phorbol esters or withdrawal of sodium butyrate increased Pgp phosphorylation while decreasing vinblastine accumulation. These studies suggest that the specificity of Pgp transport can be modulated by phosphorylation and that vinblastine, adriamycin, or actinomycin D transport, but not that of colchicine, is diminished after dephosphorylation by sodium butyrate.     

Cytosolic superoxide dismutase activity after photodynamic therapy, intracellular distribution of Photofrin II and hypericin, and P-glycoprotein localization in human colon adenocarcinoma

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered and then activated by exposure to a light source of applicable wavelength. Multidrug resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin II (Ph II) and hypericin (Hyp) on sensitive and doxorubicin-resistant colon cancer cell lines. Changes in cytosolic superoxide dismutase (SOD1) activity after PDT and the intracellular accumulation of photosensitizers in sensitive and resistant colon cancer cell lines were examined. The photosensitizers' distributions indicate that Ph II could be a potential substrate for P-gp, in contrast to Hyp. We observed an increase in SOD1 activity after PDT for both photosensitizing agents. The changes in SOD1 activity show that photodynamic action generates oxidative stress in the treated cells. P-gp appears to play a role in the intracellular accumulation of Ph II. Therefore the efficacy of PDT on multidrug-resistant cells depends on the affinity of P-gp to the photosensitizer used. The weaker accumulation of photosensitizing agents enhances the antioxidant response, and this could influence the efficacy of PDT.     

Cytomorphological lesions induced by chemotherapeutic agents in transitional cell carcinoma in vitro

Summary Transitional cell carcinoma from 20 patients and two human cell lines were maintained in short-term tissue culture. Each was studied ultrastructurally before and after incubaton with cisplatinum, adriamycin, or mitomycin C. Sequential ultrastructural changes were noted and were found to be specific for each agent tested. Ultrastructural changes in the nucleoli were produced by exposure to cisplatinum or mitomycin C; alterations in the heterochromatin of the nuclei were characteristic of treatment with adriamycin. The changes in the nucleoli seen with cisplatinum have not been described previously and support an alkylating property as a mechanism of action. Intravesical chemotherapeutic agents are now commonly used in clinical treatments. The morphological changes produced by these agents are specific and may be seen in the clinical setting. This research was supported by the Veteran's Administration Merit Review grant.