Tumor localization of newly developed hematoporphyrin (DHP) using a bladder tumor model: a novel hematoporphyrin derivative

Application of laser irradiation with porphyrin(s) or their derivatives for the destruction of tumors in humans requires preliminary studies of their localization in normal and malignant tissues. A novel derivative of hematoporphyrin (HP) was prepared. The newly developed hematoporphyrin (DHP) was administered to Fisher rats with bladder tumors and showed greater accumulation in the tumoral tissues. Comparative data on (HP) and (DHP) are presented and discussed in light of the enhanced tumor porphyrin uptake caused by these agents. The homogeneous intense fluorescence noted with DHP-treated animals suggests that total tumor kill curative therapy will be more feasible. The study paves the way to refining increased porphyrin augment phototherapy and laser application in the field of oncology.     

Preferential uptake of cytotoxic porphyrins from hematoporphyrin derivative in murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells

Photodynamically induced loss of clonogenicity of murine L929 fibroblasts and Chinese hamster ovary K1 epithelial cells was determined with two different assays. It appeared that the loss of clonogenicity was much higher when 20 cells/cm2 were incubated with hematoporphyrin derivative (HPD) and illuminated, than when confluent cell layers were incubated with the same amount of HPD and illuminated prior to plating out. This dependency of cell killing on the experimental protocol was also observed when protoporphyrin (90-95% pure) was used as photosensitizer, but not when the cells were photodynamically treated with rose bengal or exposed to mitomycin C. Further, when cell layers were incubated with the residual solution that remained after the previous incubation of a confluent cell layer with HPD, illumination of these layers appeared to be almost non-toxic, although the overall porphyrin concentration in the residual solution was only slightly lower than in HPD. These results indicate that the porphyrins, responsible for loss of clonogenicity, are present in relatively small amounts in HPD and unpurified protoporphyrin and are preferentially taken up by the cells. Although 2-aminoisobutyric acid transport and DNA synthesis are among the most photosensitive targets with HPD, photodynamic treatment of L929 cells with the residual solution did not result in inhibition of the transport system and DNA synthesis. In contrast, the K+ content of the cells still decreased considerably, when utilizing the porphyrins, remaining in the residual solution as sensitizer. This indicates that under the present experimental conditions the disturbance of the membrane barrier function does not contribute to loss of clonogenicity of these cells and, moreover, that the photodynamically induced K+ leakage is caused by a component of HPD other than inhibition of 2-aminoisobutyric acid transport and DNA synthesis.     

声化学激活血卟啉诱导艾氏腹水肿瘤细胞凋亡

本实验采用频率为2．0MHz，声强分别为1．0w／cm^2、1．5w／cm^2、2．0w／cm^2等不同参数，研究超声激活血卟啉对艾氏腹水肿瘤细胞的杀伤作用和诱导肿瘤细胞凋亡现象。通过扫描电镜、透射电镜以及荧光显微镜观察受损后细胞形态结构的变化，主要表现为细胞微绒毛的减少，胞膜结构和通透性的改变，细胞器的受损以及核物质的分解、丢失；同时发现处理后的肿瘤细胞有核物质凝集、趋边排列以及凋亡小体的形成等细胞凋亡特征。研究中首次发现声化学激活血卟啉在对艾氏腹水肿瘤细胞杀伤的同时，也能诱导艾氏腹水肿瘤细胞发生凋亡，提示在声动力疗法中并存着对癌细胞的直接杀伤和通过诱导癌细胞凋亡的两种抗癌途径。     

The effect of hematoporphyrin derivative and human erythrocyte ghost encapsulated hematoporphyrin derivative on a mouse myeloma cell line

The effect of hematoporphyrin derivative (HPD) in combination with HeNe laser radiation on a mouse myeloma cell line, P3X63Ag8U1, has been examined. The effects of varying doses of radiation and photosensitizer were assessed using both dye exclusion and clonogenic assay systems. Development of photosensitivity by those cells in the presence of hematoporphyrin (HP) and HPD has been examined. In addition, with a view towards assessing erythrocyte encapsulation as a vehicle for an immunotargeting system for HPD, the effect of HPD encapsulated in such a manner, on the mouse myeloma cell line has been examined. From the results obtained from these in vitro experiments the authors believe that this may be a convenient means of packaging HPD for antibody-mediated delivery to tumour cells.     

Pharmacokinetic studies of hematoporphyrin derivatives on rat hepatocytes. Protoporphyrin dimethyl ester hematoporphyrin ether versus dihematoporphyrin ether-free hematoporphyrin derivative

Rat liver cells incorporate monomeric as well as dimeric hematoporphyrin derivatives. Time-dependent incubation assays gave evidence that monomeric compounds are more efficiently incorporated compared to protoporphyrin dimethyl ester hematoporphyrin ether. HL60 cells take up dimeric porphyrins in substantially higher quantities than hepatocytes do. These results allow the conclusion that physiological versus tumor cells behave differently with respect to porphyrin uptake: Whereas physiological cells prefer monomeric porphyrins, tumor cells preferentially incorporate dimeric porphyrins.     

On the preparation of dihematoporphyrin ether-free hematoporphyrin derivative

A dihematoporphyrin ether-free hematoporphyrin derivative has been prepared by a base-catalysed dehydration of hematoporphyrin with sodium hydroxide. The identification was performed by HPLC and mass spectroscopy (FD-MS). The reaction of hematoporphyrin with 1 M sodium hydroxide for 24 h yields more than 90% of the monomeric porphyrins.     

Studies of hematoporphyrin and hematoporphyrin derivative equilibria in heterogeneous systems. Porphyrin-liposome binding and porphyrin aqueous dimerization

Two processes of porphyrins in heterogeneous systems containing aqueous and membrane phases have been studied with hematoporphyrin and hematoporphyrin derivative: Dimerization equilibrium in the aqueous phases and porphyrin-membrane binding equilibrium using liposomes as models for biological membranes. The interrelationship of aqueous aggregations and membrane binding was probed and the porphyrin aggregation state in the membrane, at equilibrium, was assessed. Fluorimetric techniques were employed. The dimerization equilibrium constants, at neutral pH and 37°C were found to be 2.8 · 10⁵ M⁻¹ and 1.9 · 10⁶ M⁻¹ for hematoporphyrin and its derivative, respectively. Over a porphyrin concentration range going from monomer-dominant to dimer-dominant systems, we have found that only monomers are bound to the membrane. The respective monomer-liposome binding constants, found to be independent of the initial monomer/dimer distribution in the aqueous phase, were determined to be 1.6 · 10³ M⁻¹ and 4.1 · 10³ M⁻¹ at neutral pH and 37°C for hematoporphyrin and its derivative, respectively. The monomer-liposome interaction was found to perurb the initial monomer/dimer distribution in the aqueous phase, so that the monomers residing at equilibrium in the membrane originate from both monomers and dimers in the aqueous phase.     

Effect of photoactivated hematoporphyrin derivative on the viability ofStaphylococcus aureus

The photodynamic effect of hematoporphyrin derivative (HPD) on the viability of penicillin-resistantStaphylococcus aureus is described. Growth rate of the bacteria was markedly reduced by exposure to light and HPD.Staphylococcus viability was decreased by 80% in 3 h of growth even at low HPD concentration (12 g/ml). A synergistic killing effect of HPD, light, and penicillin (10 g/ml) onStaphylococcus aureus was demonstrated, although the bacteria were originally resistant to 100 g/ml penicillin. A residual viability of only 3% was found by growth in medium containing this drug combination. The surviving bacteria after 3 h of treatment were sensitive even to 1 g penicillin/ml. The mechanism of HPD action onStaphylococcus is composed of two steps: i) penetration of HPD into the bacterial cell, which may be accomplished in the dark with no harm to the cells; ii) damaging of the bacterial cell upon photoactivation. The photoactivated HPD completely inhibited thymidine incorporation into the bacterial DNA. This effect was accompanied by inhibition of RNA and protein synthesis, which was parallel in extent to the reduction in growth rate. Electron microscopic examination ofS. aureus exposed for 3 h to HPD and light showed the appearance of well-developed mesosomes in the bacterial cells. None of these effects was observed on Gram-negative bacteria.     

Immunological enhancement of a murine allogeneic tumor in absence of the spleen.

An AKR lymphoma, conditioned to grow in BALB mice by inoculating it within a subcutaneously implanted glass cylinder, led to the development of an allogeneic tumor, lymphoma P, in 65% of the animals killing them in an average of 39 days. Splenectomy performed 12 days before tumor challenge significantly decreased the incidence of lymphoma P to 40%. However, the condition of maximal tumor enhancement obtained by pretreatment of the host with soluble tumor antigen 10 days before tumor challenge remained unaltered by splenectomy, 91% of the animals dying of tumor as compared to 92% of the controls.     

In vivo assessment of 111in-labeled hematoporphyrin derivative in breast tumor-bearing animals

The biological behavior of ¹¹¹In-labeled HPD has been investigated in tumor-bearing animals. Mice mammary adenocarcinomas and 7,12-dimethylbenz(a)anthracine induced breast tumors in Sprague-Dawley female rats were clearly visualized by ¹¹¹In-HPD nuclear scintigraphy. Optimal scans were obtained after a 48 h delay. In normal and tumor-bearing animals, the highest uptake of ¹¹¹In-HPD 72 h post-injection was found in the liver, the spleen and the kidneys. Depending on the size and the extent of necrosis, the uptake of ¹¹¹In-HPD by malignant breast tumors varied from 2.5% injected dose (ID) (range 0.14–5.3% ID) in mice to 1% ID (range 0.22–8.1% ID) in rats. Benign breast tumor uptake of ¹¹¹In-HPD was less that 1%ID. No significant amount of the radiopharmaceutical was found in pulmonary abscesses and abdominal cysts (< 0.1 % ID). Scintigrams of these infectious or inflammatory lesions were normal. Malignant tumor to blood, heart and lung ratios averaged 50:1, 10:1 and 3:1 respectively. Tumor to brain ratio ranged from 72 to 444:1.     

声化学诱导艾氏腹水瘤细胞凋亡机制初探

本研究采用频率1.43MHz,声强3W/cm2的高频聚焦超声处理艾氏腹水肿瘤细胞,研究超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡的途径及其与癌细胞内的氧自由基之间的关系。通过细胞免疫组织化学方法检测与癌细胞凋亡相关的Bax,细胞色素c和caspase-3蛋白的动态表达,黄嘌呤氧化酶法检测超氧化物歧化酶活性变化,硫代巴比妥酸法检测膜脂质过氧化物的含量。结果发现超声加血卟啉处理1h,癌细胞胞浆中的三种促凋亡蛋白表达增多,3h时表现为高表达;处理1h的癌细胞,超氧化物歧化酶活性下降,膜脂质过氧化物增多。研究结果表明超声激活血卟啉诱导艾氏腹水肿瘤细胞凋亡可能通过线粒体途径,且与癌细胞受损后产生的氧自由基有关。     

Galactosyltransferase activities in cultured urinary bladder tumor cells

After demonstrating that three bladder cancer cell lines (human bladder transitional cell carcinoma, MGH-U1; rat bladder transitional cell carcinoma, RBTCC; Nara rat bladder epidermal carcinoma, NBT-II) had galactosyltransferase (GT) activity in their cell surfaces, we investigated the effect of increasing cell density on the activity of this enzyme. All three cell lines responded to increased cell density by increased activity of cell-surface GT towards endogenous acceptor. By the use of exogenous acceptor, we showed that in the two transitional cell carcinoma lines (human and rat), the increased activity was probably caused by increased levels of endogenous acceptor rather than enzyme. In the rat bladder epidermal carcinoma line, on the other hand, increased GT activity seemed to be the result of increased levels of the enzyme. These conclusions were supported by the increased shedding of GT into the medium with increasing cell density in case of the epidermal carcinoma cells, but not the two transitional cell carcinoma lines. Total cell-associated GT activity would indicate that, in contrast to the two transitional cell carcinoma lines, the bladder epidermal carcinoma cells may have an increased rate of synthesis of GT as confluence is approached.     

Efficient enhancement of lentiviral transduction efficiency in murine spermatogonial stem cells

Spermatogonial stem cells (SSCs) are the foundation of spermatogenesis throughout postnatal life in male and have the ability to transmit genetic information to the subsequent generation. In this study, we have optimized the transduction efficiency of SSCs using a lentiviral vector by considering different multiplicity of infection (MOI), duration of infection, presence or absence of feeder layer and polycationic agents. We tested MOI of 5, 10 or 20 and infection duration of 6, 9 or 12 h respectively. After infection, cells were cultured for 1 week and as a result, the number of transduced SSCs increased significantly for MOI of 5 and 10 with 6 h of infection. When the same condition (MOI of 5 with 6 hours) was applied in presence or absence of STO feeder layer and infected SSCs were cultured for 3 weeks on the STO feeder layer, a significant increase in the number of transduced cells was observed for without the feeder layer during infection. We subsequently studied the effects of polycationic agents, polybrene and dioctadecylamidoglycyl spermine (DOGS), on the transduction efficiency. Compared with the polybrene treatment, the recovery rate of the transduced SSCs was significantly higher for the DOGS treatment. Therefore, our optimization study could contribute to the enhancement of germ-line modification of SSCs using lentiviral vectors and in generation of transgenic animals.     

Synergistic effects of focus ultrasound with different frequency and hematoporphyrin on human tumor cells

Ｐｈｏｔｏｄｙｎｅｓｔｉｃｔｈｅｒａｐｙ(ＰＤＴ)ｈａｓｂｅｅｎｅｘｔｒｅｍｅｌｙｗｅｌｌｓｔｕｄｉｅｄｓｉｎｃｅＤｏｕｇｈｅｒｔｙａｎｄｃｏｗｏｒｋｅｒｓｏｂｓｅｒｖｅｄｔｈｅｏｂｖｉｏｕｓａｎｔｉｔｕｍｏｒｅｆｆｅｃｔｏｆｔｈｅｃｏｍｂｉｎａｔｉｏｎｏｆｌａｓｅｒｗｉｔｈｈｅｍａｔｏｐｏｒｐｈｙｒｉｎ[1].Ａｎｄｉｔｈａｓｂｅｅｎｕｓｅｄｉｎｔｈｅｃｌｉｎｉｃｔｈｅｒａｐｙｆｏｒｈｕｍａｎｔｕｍｏｒｓ[2].Ｈｏｗｅｖｅｒ,ｔｈｅｌｉｇｈｔｈａｓｔｈｅｄｉｓａｄｖａｎｔａｇｅｏｆｐｏｏｒｐｅｎｅｔｒ…     

Synergistic effects of focus ultrasound with different frequency and hematoporphyrin on human tumor cells

Human hematopoietic cell K₅₆₂, human melenonla cell LiBr and human stomach cancer cells were exposed to ultrasound (US, 1.75 W/cm², 1.4, 2.16 and 2.4 MHz)in vitro in the presence or absence of hematoporphyrin (Hp, 100 μg/mL). The cell damaging effects of treatments were determined by means of the Trypan Blue dye exclusion test, MTT test and FDA test. The experimental results showed that the same cell line had different sensibilities to the US of different frequencies, and different cell line had different damage at the same acoustical radiation. The cornbined treatment with US and Hp enhanced greatly the cell damage, and no sensibility of insonation cells to US with Hp was observed. The cell damage tests showed that the results of MTT test corresponded well with that of Trypan Blue dye test. Project supported by the Natural Science Foundation of Shaanxi Province