A NEW PHOTOSENSITIZER SULFONATED ALUMINUM PHTHALOCYANINE:IN VITRO STUDY ON ITS PHOTODYNAMIC ACTION

The photosensitizing effects of sulfonated aluminum phthalocyanine on human liver cancer cells were studied by determining the kinetics of its cellular uptake, its state of aggregation therein and its photocytotoxicitic effect on these cells. Fluorescence methods were used to measure the cellular uptake in cell extracts and intact cells in monolayer. Results exhibited that the cellular uptake increases with the incubation time of sulfonated aluminum phthalocyanine (Alspc) and saturates at 24 hours. This relation was in coincidence with that between Alspc's photocytotoxic effect and the Alspc incubation time. Although the average Alspc concentration in cells is higher than the incubation concentration of Alspc, laser fluorescence experiments showed that the fluorescence peak of Alspc in cells incubated in higher concentration coincides with that of its aqueous solution of low concentration, suggesting that Alspc in cancer cells exists in monomer state. The results that lipid peroxidation in cells is enha     

PHOTODYNAMIC AND PHOTOBIOLOGICAL ACTION OF SULFONATED PHTHALOCYANINES

Zinc sulfonated phthalocyanlne (ZnSPc) 10 - 20 mg/ kg plus light showed strong inhibition on mouse S-180 and RA795 lung carcinoma, Inhibition rate was 44.8% and 57. 4% respectively. Aluminum sulfonated phthalocyanlne (AlSPc) 20 mg/kg plus copper vapor laser or red light, tumor Inhibition rate of S- 180 was 62. 7% and 66. 8% respectively, AISPc 1.25 μg/ml caused severe photohe-molyses of red cells. At the dose of 10 μg/ml plus light, lipld peroxidation of red cell membrane was greatly enhanced. Acetylchollnesterase (AchEs) of red cell membrane inactivated, liver mitochondrial ATPase and microsontal G-6-Pase Inhibited. ZnSPc at the dose of 1. 25 μg/ml caused obvious photodamage on DNA of hepatoma cells.     

THE SPECIFIC PHOTODYNAMIC EFFECTS OF MONOCLONAL ANTIBODIES CONJUGATED WITH HEMATOPORPHYRIN DERIVATIVE ON GASTRIC CANCER IN VITRO AND IN VIVO

Murine monoclonal antibody (MoAb) BB4.3, raised against the human gastric cancer cell line BGC823, was puriffied with Protein A-Sepharose CL-4B affinity chromatography and identified as IgG2a. It was then conjugated with a hematoporphyrin derivative (HPD) by using carbodiimide. The qualitative analysis of this conjugate showed that the amount of free HPD was negligible and there were no IgG aggregates among the conjugates. The conjugate retained both the antibody and photochemical activity of HPD.In vitro, the phototoxic effect of this HPD-BB4.3 conjugate on target cells was about 15 times higher than that of free HPD. The quality of selective photocytotoxicity was proven by the greater cytotoxi-city this conjugate showed than that of corresponding normal mouse IgG (NIgG) conjugated with HPD. It showed less cytotoxicity to colon cancer cell line B-80 (negative reaction to MoAb BB4.3) than to BGC825. Moreover, its cytotoxicity to BGC823 cells could be blocked specifically by excess BB4.3 antibody, but no     

INVESTIGATION OF THE THERAPEUTIC EFFECT OF EXPRESSION OF TRAIL IN VIVO ON MOUSE HEPATOCELLULAR CARCINOMA

Objective: To construct an eukaryotic expressing plasmid of mouse TRAIL (mTRAIL), and investigate its ability to induce the apoptosis of hepatocellular carcinoma cells in vitro and in vivo, its inhibitory effect on the growth of hepatocellular carcinoma, and its synergism with pCH510, an eukaryotic expressing plasmid of recombinant human FN polypeptide.Methods: The eukaryotic expressing plasmid of mTRAIL was constructed by RT-PCR and DNA recombination techniques. Gene transfection was performed in vitro and in vivo. The apoptosis rate of hepatocellular carcinoma cells was measured by Flow Cytometry. The apoptosis of hepatocellular carcinoma cells was measured by Flow Cytometry.The apoptosis of hepatocellular carcinoma cells was detected by TdT-mediated dUTP nick end labeling (TUNEL) and histochemistry techniques. The inhibitory effect of gene transfection on solid tumor was observed in mice. Results: The cDNA of mTRAIL was amplified by RT-PCR from the RNA of mouse spleen cells, and cloned into the eukaryotic expressing vector pcDNA3.1. The recombinant plasmid was designated as pX1. The BHK cells transfected with plasmid pX1 could attack H22 hepatocellular carcinoma cells and induce the apoptosis of them. The transfection of plasmid pX1 through injection into mouse muscles could inhibit the growth of hepatocellular carcinoma by inducing the apoptosis of tumor cells. Plasmid pX1 and pCH510 had a synergistic inhibitory effect on the hepatocellular carcinoma growth. Conclusion: Plamid pX1 could be expressed in cells and in vivo in mouse. The expression of pX1 in vivo and in vitro could induce the apoptosis of hepatocellular carcinoma ceils and inhibit the growth of hepatocellular carcinoma. Plasmid pX1 and pCH510 had a synergistic inhibitory effect on the hepatocellular carcinoma growth.     

ISOLATION AND CULTURE OF TUMOR-INFILTRATING LYMPHOCYTES FROM MOUSE HEPATOMA

By uaing enzyme digestion and Flcoll- Hypaque or Percoll discontinuous density methods, we have successfully obtained tumor-infiltrating lymphocytes (TIL) from mouse hepatoma. When analyzing the purity of TIL after separation. It was found that Percoll was more effective than Flcoll (P<0. 01). TIL could be activated In the presence of recombinant lL-2 (rIL-2) and begin to expand after culturing for 5-7 days, the tumor cells tend to decrease and disappeared after 14 days or so. TIL increased 105-fold over 40 days. Conditioned medium containing supernatant of PHA and rIL- 2 stimulated syngeneic spleen cell culture could promote the expansion of TIL.     

EFFECTS AND MECHANISMS OF ENDOSTATIN ON THE GROWTH OF OVARIAN CANCER SKOV3 CELLS IN VITRO AND IN VIVO

Objective： To evaluate the inhibitory effect of Endostatin on ovarian cancer cell line SKOV3 and to investigate the possible mechanism of the inhibition. Methods： Using MTT, transmission electron microscope （TEM） and immunocytochemistry, the effects of Endostatin on the proliferation of SKOV3 cells were studied. Nude mice were subcutaneously implanted with SKOV3 cells. The cell apoptosis of implanted tumor was detected by TUNEL and TEM. The expressions of bcl-2 and bax in implanted tumor tissues were measured by RT-PCR and immunohistochemistry. Results： Endostatin significantly inhibited the proliferation of SKOV3 cells in vitro （P〈0.01） and induced cell apoptosis, whereas the expressions of bcl-2 and bax were not changed obviously in SKOV3 cell treated with Endostatin. The mean tumor weight of Endostatin treated group was markedly lower than that of PBS control group （P〈0.05）. The expression of bcl-2 was down-regulated in Endostatin treated group, but bax was not influenced. Conclusions： The results demonstrated that Endostatin might have anti-tumor effect on ovarian carcinoma. One of the important mechanisms of Endostatin effect of anti-angiogenic and anti-tumor activities might involve regulating the bcl-2/bax expression and inducing apoptosis.     

EFFECTS OF SUPEROXIDE DISMUTASE ON SOME BIOLOGICAL CHARACTERISTICS OF LUNG CANCER CELLS IN VIVO AND IN VITRO

The present study observed the effects of superoxide diamutase (SOD) and its Inhibitor, diethyldithiocarbamate (DDC), on the metastasis of Lewis Lung cancer and some biological characteristics of A548 lung cancer cell line. It was found that SOD and DDC inhibited significantly the metastasis of Lewis lung cancer In C57 BL mice, which the effect of DDC was more significant than that of SOD, and decreased the proliferation of A549 lung cancer cell and its transplantation rate in nude mice.     

ANTITUMOR EFFECTS OF POLYETHYLENE GLYCOLMODIFIED RECOMBINANT HUMAN INTERLEUKIN-2 ON MOUSE UTERINE CERVICAL CARCINOMA IN VIVO

Rqcombinanthumaninterleukin-2(rIL-2)isoneofthecytokines,whichplananimportantroleinantitllmorimmunotherapy.However,rIL-2haslimitedsolubility,andrIL-2israpidlyclearedfromcirculationofhumanwhenitisinjectedintohuman,resultinginshort-termplasmahalf-lifeandlimitedclinicalapplication.WehavemodifiedrIL-2bypolyethyleneglycol(PEG),whichincreaseditssolubilityandprolongeditsplasmahalf-life.Inthispaper,theantitumoreffectsofPEG-modifiedrecombinanthumaninterleukin-2(PEG-rIL-2)againstmouseuterinecervi…     

USE OF DIFFERENT PLANT SYSTEMS IN VIVO IN COMPARING EFFECTS OF ZIRCONIUM AND THALLIUM SALTS ON CELL DIV.

In view of the wide use of plants as test systems theeffects of acute and chronic exposure were comparedon seeds of Pisum sativum and in meristematic cellsof Allium sativum root tips.The two salts comparedwere zirconium oxychloride and known to be ubiquitousin plant systems and thallium acetate known for toxiceffects in high doses.Seeds were soaked in five     

GENOTOXIC EFFECTS IN EPILEPTIC PATIENTS ON COMBINATIONS OF ANTICONVULSANT THERAPY

Sister chromatid exchange (SCE) frequency,a sensi-tive indicator in mutagenicity testing,and mitoticindex (MI) have been studied to observe genotoxiceffects in epileptic patients on routine combi-nations of anticonvulsant therapy.All patients,     

A new photosensitizer sulfonated aluminum phthalocyanine:In vitro study on its photodynamic action

The photosensitizing effects of sulfonated aluminum phthalocyanine on human liver cancer cells were studied by determining the kinetics of its cellular uptake, its state of aggregation therein and its photocytotoxicitic effect on these cells. Fluorescence methods were used to measure the cellular uptake in cell extracts and intact cells in monolayer. Results exhibited that the cellular uptake increases with the incubation time of sulfonated aluminum phthalocyanine (Alspc) and saturates at 24 hours. This relation was in coincidence with that between Alspc’s photocytotoxic effect and the Alspc incubation time. Although the average Alspc concentration in cells is higher than the incubation concentration of Alspc, laser fluorescence experiments showed that the fluorescence peak of Alspc in cells incubated in higher concentration coincides with that of its aqueous solution of low concentration, suggesting that Alspc in cancer cells exists in monomer state. The results that lipid peroxidation in cells is enhanced by Alspc photosensitization reflected that it may be one of the mechanisms of cell damage. The photodamage on cells was also studied with 3T3 mouse cells (conversion), showing agreeable results to that with liver cancer cells, which suggests that Alspc’s photocytotoxic effect is nonselective to cell types.     

DNA damage produced by combined hyperglycemia and hyperthermia in two mouse fibrosarcoma tumors in vivo

In this study we used alkaline elution to examine DNA damage produced in two murine fibrosarcomas after hyperthermia (42°C), with or without preinduced hyperglycemia. The work was stimulated by a recent report that pretreatment of mice with glucose prior to hyperthermin decreased the growth rate in a similar fibrosarcoma tumor. The intercellular tumor pH dropped from its resting value of 7.1 to a value of 6.6 at 1.5 hr after a single injection of glucose. While treatment with either glucose alone or heat alone produced very little detectable damage, the combination of these two agents resulted in marked degradation of tumor DNA isolated immediately after treatment. DNA degradation was accompanied by a simultaneous decrease in cell viability; thus, direct cell killing and subsequent nuclease or lysosomal enzyme activity are probably involved. We also tested whether hyperglycemia combined with hyperthermia influenced the DNA-DNA crosslinking induced by subsequent cyclophosphamide (Cy) treatment. Because of the extensive degradation caused by the pretreatment alone under the conditions used in these initial experiments, we were not able to quantitate with validity the amount of Cy-induced crosslinking. However, damage in viable cells may presumably interact with the subsequent Cy treatment to produce further selective cell killing in the tumor.     

Comparison of aluminium (III) phthalocyanine tetrasulfonate- and meta-tetrahydroxyphenylchlorin-monoclonal antibody conjugates for their efficacy in photodynamic therapy in vitro

A challenge in photodynamic therapy (PDT) is to improve the tumour selectivity of the photosensitizers by using monoclonal antibodies (MAbs). With this aim, we developed MAb-conjugates with the hydrophobic photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) and with the hydrophilic sensitizer aluminium (III) phthalocyanine tetrasulfonate (AlPcS(4)). The capacity of these photoimmunoconjugates for selective targeting of squamous cell carcinoma (SCC) in vivo was demonstrated previously in SCC-bearing nude mice. Preliminary in vitro PDT studies with the vulvar SCC cell line A431 showed promising phototoxicity with both sensitizers when coupled to the internalizing MAb 425. To rank the photosensitizers for their potential in photoimmunotherapy, we herein describe an extensive in vitro evaluation of mTHPC-MAb and AlPcS(4)-MAb conjugates. Both classes of conjugates were directly compared using 5 different SCC cell lines as target and 3 different MAbs (BIWA 4, E48 and 425) for tumour cell targeting. In contrast to free AlPcS(4) (IC(50) > or = 700 nM), MAb-conjugated AlPcS(4) was found to be highly phototoxic in PDT in all 5 cell lines. AlPcS(4)-BIWA 4 was most consistently effective with IC(50) values ranging from 0.06-5.4 nM. mTHPC-MAb conjugates were in general hardly effective. Phototoxicity (log IC(50)) of the AlPcS(4)-MAb conjugates was found to be strongly correlated with their total cell binding capacity (internalized and surface bound) and to be less correlated with their internalization capacity. In conclusion, these data show a high potential of AlPcS(4)-MAb conjugates in comparison to mTHPC-MAb conjugates for use in PDT.     

The significance of thermotolerance after 41 degrees C hyperthermia: in vivo and in vitro tumor and normal tissue investigations

We have investigated the development of thermotolerance in both tumors and normal tissues after 41 degrees C for durations as brief as 15 minutes. The murine RIF tumor, treated by both local radiofrequency and systemic methods, was assayed for thermotolerance by both tumor growth and cell survival analyses. The murine leg and ear, treated by conductive methods, were assayed using pre-defined tissue damage scoring systems. All of these treatments quickly induced substantial levels of thermotolerance. In the tumor studies using local heating, RIF mean diameter doubling time decreased from 17.8 days to a minimum of 13.0 days with a 9 hr interval between 41.0 degrees C for 15 minutes and 44.0 degrees C for 30 minutes (9 hr D1-D2); cell survival increased from 1.2 X 10(-2) to 3.4 X 10(-1) (same interval). Both assays showed some degree of tolerance present immediately after 41.0 degrees C for 15, 30 or 60 minutes (0 hr D1-D2). In the tumor studies using systemic heating, the kinetic pattern of the induced tolerance was similar to that observed after local heating. In the leg studies, 41.0 degrees for 30 minutes increased the time at 45 degrees C necessary to induce a specified level of tissue damage (mean score of 7) by a maximum of 1.8 times (24 hr D1-D2). The kinetic pattern was similar to that for the tumors. In the ear studies, 41.0 degrees C for 30 minutes increased the time at 45 degrees C necessary to induce ear necrosis in 50% of animals by a maximum of 3.5 times (48 hr D1-D2). The peak tolerance level occurred later for the ears, which have a lower normal temperature of 28-30 degrees C, than for the tumors or legs. These results indicate that: thermotolerance can begin to appear in tumors during treatment if hyperthermia sessions involve initial low thermal exposures (near 41 degrees C) for 15 minutes or longer; thermotolerance can develop in tumors after systemic heating and occurs with a kinetic pattern similar to that following local heating; and normal tissues also can develop high levels of thermotolerance after similar thermal exposures.     

The in vivo interaction between flavone acetic acid and hyperthermia

The in vivo interaction between flavone acetic acid (FAA) and hyperthermia was studied in a C3H mammary carcinoma grown in the feet of female CDFl mice and in normal foot skin. FAA was intraperitoneally injected prior to local tissue heating in restrained non-anaesthetized animals. Alone, FAA at doses of 100 mg/kg and above, inhibited tumour growth in a dose-dependent fashion. FAA also enhanced the tumour response to heat, the effect being dependent on both the time interval between the two modalities and the FAA dose, the greatest effect occurring when FAA doses of ≥ 150 mg/kg preceeded heat by 3–48h. These effects of FAA correlated with the drug's ability to decrease tumour blood perfusion measured using the RbCl extraction procedure. Injecting 150 mg/kg FAA 3h before heating (42.7d`C) resulted in a 2–2-fold increase in tumour heat damage, but had little effect on the response of normal foot skin in non-tumour-bearing mice. However, this treatment gave a 20-fold increase in normal tissue damage when the skin experiments were repeated in tumour-bearing animals. These effects in skin occurred in the absence of any blood perfusion changes, but appeared to be associated with FAA-induced TNF-α production.     

Relationship between thermal parameters and tumor response in hyperthermia combined with radiation therapy

Background. In hyperthermia for cancer therapy, thermal parameters related to tumor response have not yet been clarified. We investigated thermal parameters that could predict tumor response to hyperthermia combined with radiotherapy in locally advanced malignancies. Methods. Fifty-four patients with locally advanced malignancies who were treated by hyperthermia in combination with radiation therapy were enrolled in this study. Local hyperthermia was induced by ultrasound heating equipment for 60 min, within 30 min after irradiation, twice a week, for a total of six to ten sessions. Radiation therapy was administered with a conventional fractionation regimen, at a total dose of 40–70 Gy. Multi-point thermometry results were obtained with every 10-s temperature data acquisition. An average of seven interstitial sites per tumor was monitored for each treatment. Univariate logistic regression analysis was used to investigate the relationship between tumor response and minimum, maximum, and average intratumor temperature (Tmin, Tmax, Tav); the cumulative minutes of treatment at temperatures exceeded by 90%, 50%, and 10% of the measured intratumoral temperatures (T90, T50, T10); and cumulative minutes of the temperature that achieved above the index temperature value in the tumor center {(Cum min T(center) > T(index)}. Results. Complete and partial response rates were 32.6% and 46.2%, respectively. Univariate logistic regression analysis revealed that the temperature parameters with predictive probability were highest for Cum min T(center) > 42.5°C, followed by >42°C and >41°C; and T90. Cum min T(center) > 42.5°C was most significantly associated with complete tumor response (P < 0.001). Conclusion. These results strongly suggest that hyperthermia is a useful adjunct to radiotherapy for increasing the local control of advanced malignancies, and that Cum min T(center) > 42.5°C could be an important thermal parameter for predicting tumor response. Received: August 7, 2000 / Accepted: March 21, 2001     

Effects of the flavonoid drug Quercetin on the response of human prostate tumours to hyperthermia in vitro and in vivo

Tumour hyperthermia, although potentially a powerful therapeutic agent and radiation sensitizer, is hindered by a number of considerations including inhomogeneous heating of deep seated tumours due to energy deposition and perfusion issues. One solution is to design hyperthermia sensitizers to amplify the effects of hyperthermia, particularly at cold spots within the tumour undergoing treatment. This study examined the use of Quercetin, a flavonoid drug shown previously to antagonize the expression of HSP72 and induce apoptosis as a sensitizer of prostate cancer growth in vivo. Quercetin dose-dependently suppressed PC-3 tumour growth in vitro and in vivo. When combined in a treatment protocol with hyperthermia, quercetin drastically inhibited tumour growth and potently amplified the effects of hyperthermia on two prostate tumour types, PC-3 and DU-145 in vivo. These experiments, thus, suggest the use of Quercetin as a hyperthermia sensitizer in the treatment of prostate carcinoma.     

Schedule-dependent interaction between Doxorubicin and mTHPC-mediated photodynamic therapy in murine hepatoma in vitro and in vivo

Purpose: To evaluate cytotoxic and antitumor effects of a conventional anticancer drug Doxorubicin (Dox) and photodynamic therapy (PDT) mediated by a promising photosensitizer of second generation meta-tetra (3-hydroxyphenyl)-chlorin (mTHPC) in combination. Methods: Murine hepatoma MH-22A was used for investigation in vitro and in vivo. In vitro, the cells were incubated with 0.15 μg/ml mTHPC for 18 h and exposed to light from LED array (λ= 660±20 nm) at 0.6–2.4 kJ/m². 0.05–0.2 μg/ml Dox was administered either 24 h prior to or immediately after light exposure (Dox→PDT or PDT + Dox, respectively). The cytotoxicity was tested by staining with crystal violet. The character of the combined effect was assessed by multiple regression analysis. In vivo, the antitumor activity was estimated by monitoring the tumor volume over time, in mice transplanted subcutaneously with MH-22A and treated with Dox and/or PDT. For PDT, mice were exposed to light from diode laser (λ=650±2 nm) at 12 kJ/m² following 24 h after administration of 0.15 mg/kg mTHPC. A 3 mg/kg Dox was administered either within 15 min prior to mTHPC or within 15 min after light exposure (Dox→PDT or PDT + Dox, respectively). Results: Both in vitro and in vivo, the combination of mTHPC-mediated PDT and Dox was evaluated to be more effective than each treatment alone. In vitro, the difference between cell viability curves after photodynamic treatment as a single modality and after combination of photodynamic treatment with Dox was statistically significant under most of the applied conditions (P≤0.02). In the case of PDT + Dox, the combination had an additive character, and the sequence Dox→PDT caused a sub-additive interaction. In vivo, both regimens of combination were more effective in inhibiting tumor growth than any single treatment (P<0.09). The antitumor activity of PDT + Dox regimen was more prominent than that of Dox→PDT; however, significance of the difference was not high (P=0.08). Conclusions: These results indicate that Dox potentiates therapeutic efficacy of mTHPC-mediated PDT and vice versa, and the degree of potentiation is influenced by the combination schedule: administration of Dox immediately after light exposure is preferable to administration of Dox at 24 h prior to light exposure.     

Effectiveness of 2',2'difluorodeoxycytidine (Gemcitabine) combined with hyperthermia in rat R-1 rhabdomyosarcoma in vitro and in vivo

Purpose : To investigate the schedule-dependency of 2 ,2 difluorodeoxycytidine (dFdC, Gemcitabine) combined with hyperthermia (HT), in vitro as well as in vivo . Materials and methods : Rat R-1 rhabdomyosarcoma cells were treated with various concentrations of dFdC for 70 min, 4h and 24h. After various time intervals HT (60min at 43 C) was applied. Cell survival was determined by clonogenic assays. Female Wag/Rij rats bearing R-1 tumours on the hind limbs were treated with dFdC (20mg/kg), with locally applied HT (60min at 43 C) or with a combined treatment using different time intervals (0, 24 and 48h). Tumour growth delay (TGD) and normal tissue toxicity were assessed. Results : With dFdC alone, significant cytotoxicity was observed after a 24h-exposure. Except for the 24h-exposure, HT reduced the cytotoxicity of dFdC in simultaneous applications. An enhanced cytotoxic effect was found when HT was applied 20h after a 4h-incubation with dFdC. In vivo , HT applied 48h after dFdC-administration resulted in potentiation of the effect of dFdC with respect to TGD without an increase in toxicity. Conclusions : The efficacy of dFdC combined with HT is schedule-dependent both in vitro and in vivo . The addition of HT enhances the effectiveness of dFdC in the R-1 tumour model.     

Influence of clamping-induced ischemia and reperfusion on the response of a mouse melanoma to radiation and hyperthermia

Purpose: To study the response of a mouse melanoma to radiation and hyperthermia under acute hypoxia and reperfusion. Materials and methods: B16F1 melanoma of 100 ± 10mm³, in C57BL mouse, were locally exposed to 10 Gy gamma radiation (RT), 43°C for 30 min (HT) in a water bath, or RT followed immediately by HT, under clamping (acute hypoxia) or 1 h after reperfusion. Tumour regression, volume doubling time (VDT), growth delay (GD), apoptosis and microvascular density (MVD) were studied. Results: Under clamping, HT increased the VDT and GD to > 20 days above control and resulted in > 50% regression (PR) in all the tumours, whilst RT + HT synergistically enhanced VDT and GD. Under reperfusion, HT produced 25% PR against 16% by RT, with no increase in VDT and GD compared to RT. RT + HT significantly enhanced VDT and GD above that of RT or HT, but did not further increase PR of reperfused tumours. HT under clamping caused > 50% increase in apoptic cells over control and decreased MVD to 1/3rd of control. RT + HT further enhanced apoptotic cells to > 70% and reduced MVD to 1/6th of control. Conclusions: These results suggest that combination of radiotherapy with hyperthermia could benefit treatment of tumours with ischemia-induced acute hypoxia.