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ORIGINAL ARTICLES
Effective photodynamic therapy in drug-resistant prostate cancer cells utilizing a non-viral antitumor vector (a secondary publication) Masaya Yamauchi 1, Norihiro Honda 1, 2, Hisanao Hazama 1, Shoji Tachikawa 3, 4, Hiroyuki Nakamura 3,Yasufumi Kaneda 5, Kunio Awazu 1, 6, 7
1: Graduate School of Engineering, Osaka University 2: Institute for Academic Initiatives, Osaka University 3: Synthetic Organic Division, Chemical Resources Laboratory, Tokyo Institute of Technology 4: Department of Life Science, Faculty of Science, Gakushuin University 5: Graduate School of Medicine, Osaka University 6: Global Center for Medical Engineering and Informatics, Osaka University 7: Graduate School of Frontier Biosciences, Osaka University
Background and Aims: There is an urgent need to develop an efficient strategy for the treatment of drug-resistant prostate cancer. Photodynamic therapy (PDT), in which low incident levels of laser energy are used to activate a photosensitizer taken up by tumor cells, is expected as a novel therapy for the treatment of prostate cancer because of the minimal invasive nature of PDT. The present study was designed to assess the efficacy of a novel vector approach combined with a conventional porphyrin-based photosensitizer. Materials and Methods: Our group focused on a non-viral vector (hemagglutinating virus of Japan envelope; HVJ-E) combined with protoporphyrin IX (PpIX) lipid, termed the porphyrus envelope (PE). It has been previously confirmed that HVJ-E has drug-delivering properties and can induce cancer-specific cell death. The PE (HVJ-E contained in PpIX lipid) was developed as a novel photosensitizer. In this study, the antitumor and PDT efficacy of the PE against hormoneantagonistic human prostate cancer cells (PC-3) were evaluated. Results and Conclusions: Our results demonstrated that, under specific circumstances, PDT using the PE was very effective against PC-3 cells. A novel therapy for drug-resistant prostate cancer based on this vector approach is eagerly anticipated. Key words: drug-resistant prostate cancer • drug delivery • photodynamic therapy • Sendai virus
1. Introduction Prostate cancer is a cancer that occurs in the male-specific organ, the prostate gland. The morbidity of this cancer type increases with age, and almost all patients are over 55 years of age 1). Prostate cancer is predicted to become one of the cancers with the highest mortality rate among male cancer patients in the near future, Addressee for Correspondence: Masaya Yamauchi A1−411, 2−1 Yamadaoka, Suita, Osaka, 565 −0871, Japan TEL: +81-6-6879-4735, FAX: +81-6-6879-7363 Corresponding author: [email protected] ©2016 JMLL, Tokyo, Japan * This article is based on a study first reported in The Journal of Japan Society for Laser Surgery and Medicine, Vol. 36-1:18-24, 2015
in Japan. For early cancers, the complete resection of the prostate gland is performed and the efficacy rate is high. For advanced cancers, hormonal treatment and chemotherapy are generally the method of treatment chosen. However, in many cases the therapeutic effect of this approach is only transient. It is very common that the tumor acquires resistance to chemotherapeutic agents over time. Once the tumor becomes drug resistant, the cancer becomes extremely difficult to treat, with no effective treatments present at this time. Methods such as intermittent treatment by lengthening the time between drug administration or the switching Received date: November 7, 2014 Accepted date: February 24, 2015
Laser Therapy 25.1: 55-62
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ORIGINAL ARTICLES of drugs to be administered have been attempted but presently is not considered as a solution to the basic problem. One method that could solve this problem is thought to be photodynamic therapy (PDT). PDT is a treatment where a tumor cell-specific photosensitizer is allowed to accumulate in the target tumor cells and tumor neovasculature, which are then irradiated with low incident levels of laser energy at wavelengths specific to the photosensitizer’s action spectrum. This leads to the excitation of the photosensitizing agents which in turn leads to the production of reactive oxygen species (ROS) restricted to within the tumor cells and vasculature. The highly cytotoxic effect of the ROS selectively destroys the tumor and its vasculature which makes this treatment very minimally invasive. There are also reports that state that PDT itself has an activating effect on the immune system and manifests anti-tumoral effects 2). PDT is anticipated to be an effective method for the treatment of drug-resistant advanced prostate cancer because of its minimal invasiveness particularly since most of the patients are elderly. It can be combined with other modes of treatment and can be performed multiple times. However there are two problems surrounding PDT remaining to be solved. One is the hospitalization period for the prevention of photohypersensitivityrelated problems. The specificity of photosensitizers is not completely limited to tumor cells and photosensitizers can persist in the skin and liver after the PDT procedure. The patient must therefore be kept in a light shielded rather than a dark environment for a certain number of days 3, 4). Secondly, there are only limited types of cancers that are capable of being treated with PDT. For PDT to take effect, the first law of photochemistry must be satisfied, namely that absorption of the laser energy in the photosensitizer must occur before there can be any reaction. The penetration of light into biological tissue is dependent on the wavelength, therefore cancers that are deep within the organ and/or cancers with insufficient accumulation of photosensitizers are poor candidates for PDT where adequate treatment outcomes cannot be expected, and become unpredictable and uncontrollable. To achieve successful PDT for anticancer drug-resistant advanced prostate cancers, the resolution of these two issues is imperative. Since the selectivity or specificity of the photosensitizers is limited, researchers have been trying to develop efficient drug delivery systems (DDSs) 5, 6) to gain greater accumulation of the photosensitizers within tumor cells. There are numerous drug delivery
56
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strategies such as DDSs using the enhanced permeability and retention (EPR) effect which utilizes the exacerbated vasopermeability of tumor tissue, and DDSs using the tumor specific antigen and antibody interaction. Many are effective showing favorable treatment results. The authors focused on a DDS employing a non-virus vector (hemagglutinating virus of Japan envelope: HVJ-E) to enclose the active molecules. HVJE is an inactivated Sendai virus particle where the replicating ability of the virus is nullified by the destruction of the virus ribonucleic acid (RNA) with UV irradiation. HVJ-E is capable of delivering its enclosed molecules into a cell through membrane fusion, due to the presence of the HN and F proteins on the envelope surface 7, 8). The great advantages of DDS with HVJ-E are that it delivers the active ingredient directly to the cytoplasm and not through endocytosis; and that the accumulation of the active agent in tumor cells occurs at a much faster rate than via the normal pathways. Also recent studies have showed that HVJ-E itself has strong beneficial effect in cancer immunotherapy. Although the HVJ-E RNA is destroyed by UV light, fragments and particles of the RNA are identified by the retinoic acid-inducible gene-I (RIG-I) within the cytoplasm and lead to the production of chemokines and cytokines. HVJ-E also enhances the maturation of dendritic cells and the production of IL-6. Such reactions activate NK cells and cytotoxic T cells while suppressing suppressor T cells, all which lead to enhanced antitumor immunity 9). As for the signal pathway of RIG-I, it is known that in human cancer cells this pathway leads to the induction of apoptosis-related genes such as Noxa and TRAIL which lead to the specific apoptosis of cancer cells 10, 11). It has been reported that HVJ-E administration in Balb/c mice transplanted with mouse colon cancer cell line CT26 resulted in a 60~80% eradication of the tumor 9). Such characteristics of HVJ-E led the authors to think that PDT for prostate cancers using DDS with HVJ-E would be very effective. The authors chose protoporphyrin IX-lipid (PpIXlipid) as the photosensitizer and enclosed it in HVJ-E and created a novel photosensitizer, the “porphyrus envelope” (PE). The concept behind the PE is enhanced PDT efficacy due to the synergy between the high DDS capability of HVJ-E and the antitumor effect of HVJ-E. In a preliminary study, PDT with PE for hormone antagonistic prostate cancer cells was compared to that of 5-aminolevulinic acid (5-ALA) and PpIX-lipid and showed favorable results 12). On the other hand, no cell deaths without laser irradiation were observed.
M Yamauchi et al
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These results indicate that the originally anticipated anti-tumor effect of HVJ-E (direct effect for cancer specific cell deaths) was not observed. The anti-tumor effect of HVJ-E had been previously confirmed at the same titer. Therefore the enclosure of PpIX-lipid may have somehow affected therapeutic efficacy of PE. In order to increase the effectiveness of PE-PDT, the research into the cause of this phenomenon and improvement in the preparation and administration of PE are necessary.
2. Purpose The purpose of this study was to develop a PE which utilizes the DDS function and anti-tumor effect of HVJE and thereby to establish an effective method of PDT for anti-cancer drug-resistant prostate cancers. This study also reported on the PDT efficacy and anti-tumor effect of a preparation of PE encapsulating a smaller amount of PpIX-lipid.
3. Material and methods 3.1 Cell culture In this study, the hormone antagonistic human prostate cancer cell line PC-3 was used as the model for anticancer drug resistant prostate cancer. PC-3 is a cell with no sensitivity to hormone drug and is a highly malignant type of cancer cell. PC-3 was cultured in Dulbecco’s modified eagle medium (D-MEM, D6046, Sigma-Aldrich) with 10% fetal bovine serum (FBS, S1820, Biowest) and 5 mL of penicillin-streptomycin mixed solution (P4458, Sigma-Aldrich).
3.2 Photosensitizers In this study, the photosensitizer chosen to be encapsulated in HVJ-E was PpIX-lipid. PpIX-lipid is a photosensitizing compound where a lipid chain is attached to PpIX, a photosensitizing compound whose precursor is 5-ALA 13). As shown in Fig. 1, the PpIX-lipid structure is very similar to the structure of the lipid of the HVJ-E particle. As a result, enclosure of PpIX-lipid into HVJ-E is accomplished by simply mixing the two together and centrifuging. Moreover, PpIX is a common substance found in living organisms with low cytotoxicity, and is inexpensive. One milliliter of a 6400 HAU/mL suspension of HVJ was dispensed to a 3.5 cm diameter plate. HAU stands for hemagglutination unit and is a measure for the degree of activity of the HN protein present in HVJ-E 14). A UV-emitting device (XL-1500 UV Cross
PDT with a non-viral vector
ORIGINAL ARTICLES Linker, Spector-Linker Co.) was used to irradiate the plate with the HVJ suspension at the energy density of 99 mJ/cm2. The UV irradiation causes fragmentation of the viral RNA of HVJ and hence causing its deactivation and thus the creation of HVJ-E 10). Centrifugation (20000 g, 4°C, 10 min.) was performed and the sediment was re-suspended with Dulbecco’s phosphatebuffered saline (D-PBS: D8537, Sigma-Aldrich) to create the HVJ-E suspension. Twenty-two microliters of PpIX-lipid prepared to a concentration of 125 µM by dilution with D-PBS was added to the 800 HAU/152 µL of HVJ-E and was warmed for 5 minutes at 37°C. Centrifugation (20000 g, 10 min.) encapsulated the PpIX-lipid into HVJ-E and the sediment was taken as the porphyrus envelope (PE) 12). Under these conditions of preparation, the encapsulated amount of PpIXlipid was 2.2 pmol/HAU per PE and in order to change the encapsulated amount of PpIX-lipid, the concentration of the PpIX-lipid was adjusted.
3.3 Comparison of the PDT effect between PpIXlipid and PE. A 96 well plate was coated with poly-L lysine prepared at a concentration of 10% (v/v) with D-PBS. PC-3 cells prepared at a concentration of 5000 cells/mL were seeded onto the wells at an amount of 100 µL/well. Twenty-four hours after incubation, 40 µL of PpIX-lipid solution was added to the wells so that the concentration of the PpIX-lipid would range from 0 to 30 µM. Forty microliters of PE (encapsulated amount of PpIXlipid: 2.2, or 0.35 pmol/HAU) was added to the well so that the concentration of PpIX-lipid would range from 0 to 1.1 µM. Three hours later, the solutions were changed to a serum-free medium and a diode laser (VLM500, Sumitomo electric industries, Ltd.) with the wavelength of 405 nm, was used for irradiation at the power density of 100 mW/cm2 for 60 sec. Since this study was targeting cells in-vitro, there is no need to compensate for light attenuation due to light-tissue interactions such as absorbance and scattering. Therefore the wavelength of 405 nm, which has a PpIX
Fig. 1: Structural formula of PpIX lipid. 57
ORIGINAL ARTICLES absorbance 20 times greater than at 630 nm, was selected. Following the irradiation, the culture medium was changed to a serum containing medium and was placed in an incubator. At 24 h or 48 h, 10 µL of a reagent for the measurement of the number of living cells, SF (07553, Nacalai Tesque), was added and 1 h later, an absorbance microplate reader (Versa MAX, Molecular Devices) was used to measure the absorbance at the wavelength of 450 nm. The absorbance of the wells with no photosensitizer solutions added was used as the control, and the proportion of the absorbance of each well was used to determine the cell survival rate.
3.4 Evaluation of the anti-tumor effect of HVJ-E and PE. A 96-well plate was coated with poly-L lysine prepared at a concentration of 10% (v/v) with D-PBS. PC3 cells prepared at a concentration of 5000 cells/mL were seeded onto the wells at the amount of 100 µL/well. After 24 h, serum containing medium or serum-free medium was used to adjust the concentration of the HVJ-E and PE to 32 HAU/mL. Thirty microliters of the solution was added to the wells and after 3 h, the media of those wells using a serum-free medium were changed to serum containing medium. Twenty-four or 48 hours later, 10 µL of the same reagent as before (SF, 07553 Nacalai Tesque) for the measurement of number of living cells was added and 1 h later, the absorbance microplate reader (Versa MAX, Molecular Devices) was used to measure the absorbance at the wavelength of 450 nm. The absorbance of the wells including the photosensitizer without irradiation was used as the control, and the proportion of the absorbance of each well was used to determine the cell survival rate.
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was centrifuged and only the erythrocytes were collected. The erythrocytes were re-suspended in D-PBS and washed. This procedure was repeated 3 times. The well-washed erythrocytes were diluted with D-PBS to reach the final concentration of 0.5 wt%. The HVJ-E and PE solutions were diluted with balanced salt solution (BSS) to a concentration minimum of 13323 fold dilution. First 50 µL of the varying dilutions of HVJ-E and PE were dispensed into the wells of a 96-well plate and 50 µL of the 0.5 wt% erythrocyte suspension were dispensed into the wells. After leaving the plate in a refrigerator for 24 hours, erythrocyte sedimentation was checked for all wells. The titer of dilution with the least HVJ-E or PE concentration with no sedimentation was defined as 1 HAU/mL and the titer of HVJ-E and PE was calculated by multiplying the dilution ratio.
4. Results. The statistical significance between data was tested
3.5 Evaluation of membrane-fusing capability of HVJ-E and PE through HA assays. In order to investigate how the amount of PpIX-lipid enclosure in HVJ-E affected the membrane-fusion capability of HVJ-E, PE with differing PpIX-lipid concentrations were prepared and evaluated. HVJ-E suspension at 6400 HAU/mL were irradiated with 99 mJ/cm2 of UV light to create HVJ-E. PpIXlipid was incorporated into HVJ-E so that the amount of PpIX-lipid per HVJ-E unit was 0, 0.04, 0.35, 0.7, 1.4, 2.2, and 17.6 pmol/HAU and thus the PE with differing PpIX-lipid concentrations were prepared. The titer of the membrane fusion was evaluated with hemagglutination (HA) assays 15, 16). The specific methods were briefly as follows. Ten milliliters of stored hen blood
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Fig. 2: Survival rate of PC-3 cells after PDT. The error bar shows a standard deviation. (a) PpIX lipid, (b) porphyrus envelope (amount of enclosed PpIX lipid in HVJ-E: 2.2 pmol/HAU).
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ORIGINAL ARTICLES
with the student’s t-test (variance unknown, unpaired, two-sided test). P values less than 0.05 were considered significant.
these conditions both HVJ-E and PE group showed a significant decrease in cell survival and the HVJ-Emediated anti-tumor effect was confirmed.
4.1 Comparison of PDT effects between PpIXlipid and PE.
4.3 Evaluation of the membrane fusion of HVJ-E and PE.
The cell survival rates after laser irradiation between PpIX-lipid and PE treatment are shown in Figure 2. In both cases cell survival decreased as the concentration of the two solutions increased and cell death due to PDT was confirmed. The half maximal inhibitory concentrations (IC50) for the two were calculated, being 1.20±0.07 µM for PpIX-lipid and 0.04±0.01 µM and 0.006±0.002 µM for PE with encapsulation rates of 2.2 pmol/HAU) and 0.35 pmol/HAU, respectively (Figure 3).
The titers of membrane fusion of PE of varying PpIX-
4.2 Evaluation of the anti-tumor effect of HVJ-E and PE. Figure 4 (a) shows the cell survival rate of PC-3 at 24 and 48 hours post-incubation after 3 hour immersion in HVJ-E or PE solutions, and after culture medium change. Even without laser irradiation, the HVJ-E group showed a significant decrease in cell survival. This confirmed the anti-tumor effect of HVJ-E on its own. However, there was only a small decrease seen for the PE group, meaning that the encapsulation of PpIX-lipid was detrimental to the anti-tumor effect of HVJ-E. Figure 4 (b) shows the cell survival rates after immersion in HVJ-E or PE for 24 or 48 hours. Under
Fig. 3: 50% inhabitant concentration of porphyrus envelope (PE) and PpIX lipid 24 h after laser irradiation. The error bar shows a standard deviation. The data of porphyrus envelope whose enclosure rate was 35 mol/ HAU was quoted from reference 12. PDT with a non-viral vector
Fig. 4: Survival rate of PC-3 cells after HVJ-E or porphyrus envelope (amount of enclosed PpIX lipid in HVJ-E: 2.2 pmol/HAU) administration without laser irradiation. The error bar shows a standard deviation. (a) 24 and 48 h incubated after 3 h immersion in HVJ-E or porphyrus envelope (It is the same condition of PDT experiment). (b) 24 and 48 h incubated with HVJ-E or porphyrus envelope.
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ORIGINAL ARTICLES lipid concentrations are shown in Figure 5. The titer of membrane fusion decreased as the concentration of PpIX-lipid increased. It was confirmed that the greater the encapsulated amount of PpIX-lipid was, the less was the membrane fusing capability of HVJ-E.
5. Discussion. 5.1 PDT effect of the porphyrus envelope. The IC50 for PE at 24 h after PDT was 0.04±0.01 µM. The same PDT effect was achieved at a concentration 1/30 of that when PpIX-lipid was administered alone. In the case of no laser irradiation, 3 hour immersion in PE solution produced a significant decrease in the cell survival rate at only 48 h incubation, as shown in Figure 4(b). This means that the main factor of the improvement in PDT efficacy is through the high accumulation of the photosensitizer into the tumor cells due to HVJ-E. Also, since the induction of PpIX-lipid to the cell is mediated through membrane fusion, PpIXlipid which is implanted on the surface of HVJ-E is considered to be re-implanted at the membrane of the cell. It has been reported that photosensitizers located in the cell membrane produce a greater PDT effect than when located in the cytoplasm 17). The localization of PpIX-lipid may have been a factor causing the difference between the PE group and the PpIX-lipid group. The cellular localization of PE and the mechanism that leads to cell death after PDT in more detail will be investigated.
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As shown in Figure 3, the IC50 of PE differed greatly depending on the enclosed amount of PpIXlipid. The reason for this is that less enclosure of PpIXlipid improved the membrane-fusion ability of HVJ-E. In earlier studies, a large amount of PpIX-lipid (44 pmol/HAU) was enclosed in a HVJ-E particle. As shown in Figure 5, an increase in the PpIX-lipid concentration led to a decrease in membrane fusion capability. This study revealed that when PpIX-lipid is enclosed at an amount of 44 pmol/HAU, PE had almost no membrane fusion capability. However, the PE solution used in this study with encapsulated PpIXlipid of 2.2 pmol/HAU, although it may not have been comparable with that of HVJ-E alone, had a membrane fusion titer of approximately 900 HAU/mL. The difference in membrane fusion titer was responsible for the drastic improvement of drug delivery efficiency which led to the improvement of the PDT effect. In actuality, this study revealed that even when the enclosed amount of PpIX-lipid was lowered to 0.35 pmol/HAU, the IC 50 was 6±2 nM, which confirmed that an improved membrane fusion titer leads to improved PDT efficacy. If only the IC50 values were used for comparison, the PpIX induction efficiency of PE with 0.35 pmol/HAU of encapsulated PpIX-lipid was 30 times greater than when PpIX-lipid was administered alone, and was 5250 times greater than the induction of PpIX by 5-ALA administration 12). In this study, laser energy at a wavelength of 405 nm was chosen for its high PpIX absorption and was used with success to achieve highly effective PDT. However, further studies with light sources delivering longer wavelengths are required in the future for in vivo studies where the light penetration depth into living biological tissues becomes a factor, since without absorption, there can be no reaction.
5.2 The anti-tumor effect of the porphyrus envelope.
Fig. 5: The titer of membrane fusion of each porphyrus envelope. The error bar shows a standard deviation.
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The direct effect of HVJ-E for the cancer specific cell death or the anti-tumor effect of HVJ-E and PE is induced by the identification of RNA fragments contained within HVJ-E, by RIG-I. In order to enhance the anti-tumor effect, the fusion of HVJ-E and PE to its targets must take place as much as possible. In this study, the anti-tumor effect of PE solution was inferior to that of HVJ-E in both cases where the tumor cells were immersed in the active solutions for 3 h and 24 h. This was thought to be due to the inferior membrane-fusing ability of PE compared to that of the original HVJ-E. When the enclosed PpIX-lipid was decreased to 0.7
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pmol/HAU, the survival rate of the cells decreased to 65% and was significantly lower (P
ORIGINAL ARTICLES
Effective photodynamic therapy in drug-resistant prostate cancer cells utilizing a non-viral antitumor vector (a secondary publication) Masaya Yamauchi 1, Norihiro Honda 1, 2, Hisanao Hazama 1, Shoji Tachikawa 3, 4, Hiroyuki Nakamura 3,Yasufumi Kaneda 5, Kunio Awazu 1, 6, 7
1: Graduate School of Engineering, Osaka University 2: Institute for Academic Initiatives, Osaka University 3: Synthetic Organic Division, Chemical Resources Laboratory, Tokyo Institute of Technology 4: Department of Life Science, Faculty of Science, Gakushuin University 5: Graduate School of Medicine, Osaka University 6: Global Center for Medical Engineering and Informatics, Osaka University 7: Graduate School of Frontier Biosciences, Osaka University
Background and Aims: There is an urgent need to develop an efficient strategy for the treatment of drug-resistant prostate cancer. Photodynamic therapy (PDT), in which low incident levels of laser energy are used to activate a photosensitizer taken up by tumor cells, is expected as a novel therapy for the treatment of prostate cancer because of the minimal invasive nature of PDT. The present study was designed to assess the efficacy of a novel vector approach combined with a conventional porphyrin-based photosensitizer. Materials and Methods: Our group focused on a non-viral vector (hemagglutinating virus of Japan envelope; HVJ-E) combined with protoporphyrin IX (PpIX) lipid, termed the porphyrus envelope (PE). It has been previously confirmed that HVJ-E has drug-delivering properties and can induce cancer-specific cell death. The PE (HVJ-E contained in PpIX lipid) was developed as a novel photosensitizer. In this study, the antitumor and PDT efficacy of the PE against hormoneantagonistic human prostate cancer cells (PC-3) were evaluated. Results and Conclusions: Our results demonstrated that, under specific circumstances, PDT using the PE was very effective against PC-3 cells. A novel therapy for drug-resistant prostate cancer based on this vector approach is eagerly anticipated. Key words: drug-resistant prostate cancer • drug delivery • photodynamic therapy • Sendai virus
1. Introduction Prostate cancer is a cancer that occurs in the male-specific organ, the prostate gland. The morbidity of this cancer type increases with age, and almost all patients are over 55 years of age 1). Prostate cancer is predicted to become one of the cancers with the highest mortality rate among male cancer patients in the near future, Addressee for Correspondence: Masaya Yamauchi A1−411, 2−1 Yamadaoka, Suita, Osaka, 565 −0871, Japan TEL: +81-6-6879-4735, FAX: +81-6-6879-7363 Corresponding author: [email protected] ©2016 JMLL, Tokyo, Japan * This article is based on a study first reported in The Journal of Japan Society for Laser Surgery and Medicine, Vol. 36-1:18-24, 2015
in Japan. For early cancers, the complete resection of the prostate gland is performed and the efficacy rate is high. For advanced cancers, hormonal treatment and chemotherapy are generally the method of treatment chosen. However, in many cases the therapeutic effect of this approach is only transient. It is very common that the tumor acquires resistance to chemotherapeutic agents over time. Once the tumor becomes drug resistant, the cancer becomes extremely difficult to treat, with no effective treatments present at this time. Methods such as intermittent treatment by lengthening the time between drug administration or the switching Received date: November 7, 2014 Accepted date: February 24, 2015
Laser Therapy 25.1: 55-62
55
ORIGINAL ARTICLES of drugs to be administered have been attempted but presently is not considered as a solution to the basic problem. One method that could solve this problem is thought to be photodynamic therapy (PDT). PDT is a treatment where a tumor cell-specific photosensitizer is allowed to accumulate in the target tumor cells and tumor neovasculature, which are then irradiated with low incident levels of laser energy at wavelengths specific to the photosensitizer’s action spectrum. This leads to the excitation of the photosensitizing agents which in turn leads to the production of reactive oxygen species (ROS) restricted to within the tumor cells and vasculature. The highly cytotoxic effect of the ROS selectively destroys the tumor and its vasculature which makes this treatment very minimally invasive. There are also reports that state that PDT itself has an activating effect on the immune system and manifests anti-tumoral effects 2). PDT is anticipated to be an effective method for the treatment of drug-resistant advanced prostate cancer because of its minimal invasiveness particularly since most of the patients are elderly. It can be combined with other modes of treatment and can be performed multiple times. However there are two problems surrounding PDT remaining to be solved. One is the hospitalization period for the prevention of photohypersensitivityrelated problems. The specificity of photosensitizers is not completely limited to tumor cells and photosensitizers can persist in the skin and liver after the PDT procedure. The patient must therefore be kept in a light shielded rather than a dark environment for a certain number of days 3, 4). Secondly, there are only limited types of cancers that are capable of being treated with PDT. For PDT to take effect, the first law of photochemistry must be satisfied, namely that absorption of the laser energy in the photosensitizer must occur before there can be any reaction. The penetration of light into biological tissue is dependent on the wavelength, therefore cancers that are deep within the organ and/or cancers with insufficient accumulation of photosensitizers are poor candidates for PDT where adequate treatment outcomes cannot be expected, and become unpredictable and uncontrollable. To achieve successful PDT for anticancer drug-resistant advanced prostate cancers, the resolution of these two issues is imperative. Since the selectivity or specificity of the photosensitizers is limited, researchers have been trying to develop efficient drug delivery systems (DDSs) 5, 6) to gain greater accumulation of the photosensitizers within tumor cells. There are numerous drug delivery
56
available at www.jstage.jst.go.jp/browse/islsm
strategies such as DDSs using the enhanced permeability and retention (EPR) effect which utilizes the exacerbated vasopermeability of tumor tissue, and DDSs using the tumor specific antigen and antibody interaction. Many are effective showing favorable treatment results. The authors focused on a DDS employing a non-virus vector (hemagglutinating virus of Japan envelope: HVJ-E) to enclose the active molecules. HVJE is an inactivated Sendai virus particle where the replicating ability of the virus is nullified by the destruction of the virus ribonucleic acid (RNA) with UV irradiation. HVJ-E is capable of delivering its enclosed molecules into a cell through membrane fusion, due to the presence of the HN and F proteins on the envelope surface 7, 8). The great advantages of DDS with HVJ-E are that it delivers the active ingredient directly to the cytoplasm and not through endocytosis; and that the accumulation of the active agent in tumor cells occurs at a much faster rate than via the normal pathways. Also recent studies have showed that HVJ-E itself has strong beneficial effect in cancer immunotherapy. Although the HVJ-E RNA is destroyed by UV light, fragments and particles of the RNA are identified by the retinoic acid-inducible gene-I (RIG-I) within the cytoplasm and lead to the production of chemokines and cytokines. HVJ-E also enhances the maturation of dendritic cells and the production of IL-6. Such reactions activate NK cells and cytotoxic T cells while suppressing suppressor T cells, all which lead to enhanced antitumor immunity 9). As for the signal pathway of RIG-I, it is known that in human cancer cells this pathway leads to the induction of apoptosis-related genes such as Noxa and TRAIL which lead to the specific apoptosis of cancer cells 10, 11). It has been reported that HVJ-E administration in Balb/c mice transplanted with mouse colon cancer cell line CT26 resulted in a 60~80% eradication of the tumor 9). Such characteristics of HVJ-E led the authors to think that PDT for prostate cancers using DDS with HVJ-E would be very effective. The authors chose protoporphyrin IX-lipid (PpIXlipid) as the photosensitizer and enclosed it in HVJ-E and created a novel photosensitizer, the “porphyrus envelope” (PE). The concept behind the PE is enhanced PDT efficacy due to the synergy between the high DDS capability of HVJ-E and the antitumor effect of HVJ-E. In a preliminary study, PDT with PE for hormone antagonistic prostate cancer cells was compared to that of 5-aminolevulinic acid (5-ALA) and PpIX-lipid and showed favorable results 12). On the other hand, no cell deaths without laser irradiation were observed.
M Yamauchi et al
available at www.jstage.jst.go.jp/browse/islsm
These results indicate that the originally anticipated anti-tumor effect of HVJ-E (direct effect for cancer specific cell deaths) was not observed. The anti-tumor effect of HVJ-E had been previously confirmed at the same titer. Therefore the enclosure of PpIX-lipid may have somehow affected therapeutic efficacy of PE. In order to increase the effectiveness of PE-PDT, the research into the cause of this phenomenon and improvement in the preparation and administration of PE are necessary.
2. Purpose The purpose of this study was to develop a PE which utilizes the DDS function and anti-tumor effect of HVJE and thereby to establish an effective method of PDT for anti-cancer drug-resistant prostate cancers. This study also reported on the PDT efficacy and anti-tumor effect of a preparation of PE encapsulating a smaller amount of PpIX-lipid.
3. Material and methods 3.1 Cell culture In this study, the hormone antagonistic human prostate cancer cell line PC-3 was used as the model for anticancer drug resistant prostate cancer. PC-3 is a cell with no sensitivity to hormone drug and is a highly malignant type of cancer cell. PC-3 was cultured in Dulbecco’s modified eagle medium (D-MEM, D6046, Sigma-Aldrich) with 10% fetal bovine serum (FBS, S1820, Biowest) and 5 mL of penicillin-streptomycin mixed solution (P4458, Sigma-Aldrich).
3.2 Photosensitizers In this study, the photosensitizer chosen to be encapsulated in HVJ-E was PpIX-lipid. PpIX-lipid is a photosensitizing compound where a lipid chain is attached to PpIX, a photosensitizing compound whose precursor is 5-ALA 13). As shown in Fig. 1, the PpIX-lipid structure is very similar to the structure of the lipid of the HVJ-E particle. As a result, enclosure of PpIX-lipid into HVJ-E is accomplished by simply mixing the two together and centrifuging. Moreover, PpIX is a common substance found in living organisms with low cytotoxicity, and is inexpensive. One milliliter of a 6400 HAU/mL suspension of HVJ was dispensed to a 3.5 cm diameter plate. HAU stands for hemagglutination unit and is a measure for the degree of activity of the HN protein present in HVJ-E 14). A UV-emitting device (XL-1500 UV Cross
PDT with a non-viral vector
ORIGINAL ARTICLES Linker, Spector-Linker Co.) was used to irradiate the plate with the HVJ suspension at the energy density of 99 mJ/cm2. The UV irradiation causes fragmentation of the viral RNA of HVJ and hence causing its deactivation and thus the creation of HVJ-E 10). Centrifugation (20000 g, 4°C, 10 min.) was performed and the sediment was re-suspended with Dulbecco’s phosphatebuffered saline (D-PBS: D8537, Sigma-Aldrich) to create the HVJ-E suspension. Twenty-two microliters of PpIX-lipid prepared to a concentration of 125 µM by dilution with D-PBS was added to the 800 HAU/152 µL of HVJ-E and was warmed for 5 minutes at 37°C. Centrifugation (20000 g, 10 min.) encapsulated the PpIX-lipid into HVJ-E and the sediment was taken as the porphyrus envelope (PE) 12). Under these conditions of preparation, the encapsulated amount of PpIXlipid was 2.2 pmol/HAU per PE and in order to change the encapsulated amount of PpIX-lipid, the concentration of the PpIX-lipid was adjusted.
3.3 Comparison of the PDT effect between PpIXlipid and PE. A 96 well plate was coated with poly-L lysine prepared at a concentration of 10% (v/v) with D-PBS. PC-3 cells prepared at a concentration of 5000 cells/mL were seeded onto the wells at an amount of 100 µL/well. Twenty-four hours after incubation, 40 µL of PpIX-lipid solution was added to the wells so that the concentration of the PpIX-lipid would range from 0 to 30 µM. Forty microliters of PE (encapsulated amount of PpIXlipid: 2.2, or 0.35 pmol/HAU) was added to the well so that the concentration of PpIX-lipid would range from 0 to 1.1 µM. Three hours later, the solutions were changed to a serum-free medium and a diode laser (VLM500, Sumitomo electric industries, Ltd.) with the wavelength of 405 nm, was used for irradiation at the power density of 100 mW/cm2 for 60 sec. Since this study was targeting cells in-vitro, there is no need to compensate for light attenuation due to light-tissue interactions such as absorbance and scattering. Therefore the wavelength of 405 nm, which has a PpIX
Fig. 1: Structural formula of PpIX lipid. 57
ORIGINAL ARTICLES absorbance 20 times greater than at 630 nm, was selected. Following the irradiation, the culture medium was changed to a serum containing medium and was placed in an incubator. At 24 h or 48 h, 10 µL of a reagent for the measurement of the number of living cells, SF (07553, Nacalai Tesque), was added and 1 h later, an absorbance microplate reader (Versa MAX, Molecular Devices) was used to measure the absorbance at the wavelength of 450 nm. The absorbance of the wells with no photosensitizer solutions added was used as the control, and the proportion of the absorbance of each well was used to determine the cell survival rate.
3.4 Evaluation of the anti-tumor effect of HVJ-E and PE. A 96-well plate was coated with poly-L lysine prepared at a concentration of 10% (v/v) with D-PBS. PC3 cells prepared at a concentration of 5000 cells/mL were seeded onto the wells at the amount of 100 µL/well. After 24 h, serum containing medium or serum-free medium was used to adjust the concentration of the HVJ-E and PE to 32 HAU/mL. Thirty microliters of the solution was added to the wells and after 3 h, the media of those wells using a serum-free medium were changed to serum containing medium. Twenty-four or 48 hours later, 10 µL of the same reagent as before (SF, 07553 Nacalai Tesque) for the measurement of number of living cells was added and 1 h later, the absorbance microplate reader (Versa MAX, Molecular Devices) was used to measure the absorbance at the wavelength of 450 nm. The absorbance of the wells including the photosensitizer without irradiation was used as the control, and the proportion of the absorbance of each well was used to determine the cell survival rate.
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was centrifuged and only the erythrocytes were collected. The erythrocytes were re-suspended in D-PBS and washed. This procedure was repeated 3 times. The well-washed erythrocytes were diluted with D-PBS to reach the final concentration of 0.5 wt%. The HVJ-E and PE solutions were diluted with balanced salt solution (BSS) to a concentration minimum of 13323 fold dilution. First 50 µL of the varying dilutions of HVJ-E and PE were dispensed into the wells of a 96-well plate and 50 µL of the 0.5 wt% erythrocyte suspension were dispensed into the wells. After leaving the plate in a refrigerator for 24 hours, erythrocyte sedimentation was checked for all wells. The titer of dilution with the least HVJ-E or PE concentration with no sedimentation was defined as 1 HAU/mL and the titer of HVJ-E and PE was calculated by multiplying the dilution ratio.
4. Results. The statistical significance between data was tested
3.5 Evaluation of membrane-fusing capability of HVJ-E and PE through HA assays. In order to investigate how the amount of PpIX-lipid enclosure in HVJ-E affected the membrane-fusion capability of HVJ-E, PE with differing PpIX-lipid concentrations were prepared and evaluated. HVJ-E suspension at 6400 HAU/mL were irradiated with 99 mJ/cm2 of UV light to create HVJ-E. PpIXlipid was incorporated into HVJ-E so that the amount of PpIX-lipid per HVJ-E unit was 0, 0.04, 0.35, 0.7, 1.4, 2.2, and 17.6 pmol/HAU and thus the PE with differing PpIX-lipid concentrations were prepared. The titer of the membrane fusion was evaluated with hemagglutination (HA) assays 15, 16). The specific methods were briefly as follows. Ten milliliters of stored hen blood
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Fig. 2: Survival rate of PC-3 cells after PDT. The error bar shows a standard deviation. (a) PpIX lipid, (b) porphyrus envelope (amount of enclosed PpIX lipid in HVJ-E: 2.2 pmol/HAU).
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ORIGINAL ARTICLES
with the student’s t-test (variance unknown, unpaired, two-sided test). P values less than 0.05 were considered significant.
these conditions both HVJ-E and PE group showed a significant decrease in cell survival and the HVJ-Emediated anti-tumor effect was confirmed.
4.1 Comparison of PDT effects between PpIXlipid and PE.
4.3 Evaluation of the membrane fusion of HVJ-E and PE.
The cell survival rates after laser irradiation between PpIX-lipid and PE treatment are shown in Figure 2. In both cases cell survival decreased as the concentration of the two solutions increased and cell death due to PDT was confirmed. The half maximal inhibitory concentrations (IC50) for the two were calculated, being 1.20±0.07 µM for PpIX-lipid and 0.04±0.01 µM and 0.006±0.002 µM for PE with encapsulation rates of 2.2 pmol/HAU) and 0.35 pmol/HAU, respectively (Figure 3).
The titers of membrane fusion of PE of varying PpIX-
4.2 Evaluation of the anti-tumor effect of HVJ-E and PE. Figure 4 (a) shows the cell survival rate of PC-3 at 24 and 48 hours post-incubation after 3 hour immersion in HVJ-E or PE solutions, and after culture medium change. Even without laser irradiation, the HVJ-E group showed a significant decrease in cell survival. This confirmed the anti-tumor effect of HVJ-E on its own. However, there was only a small decrease seen for the PE group, meaning that the encapsulation of PpIX-lipid was detrimental to the anti-tumor effect of HVJ-E. Figure 4 (b) shows the cell survival rates after immersion in HVJ-E or PE for 24 or 48 hours. Under
Fig. 3: 50% inhabitant concentration of porphyrus envelope (PE) and PpIX lipid 24 h after laser irradiation. The error bar shows a standard deviation. The data of porphyrus envelope whose enclosure rate was 35 mol/ HAU was quoted from reference 12. PDT with a non-viral vector
Fig. 4: Survival rate of PC-3 cells after HVJ-E or porphyrus envelope (amount of enclosed PpIX lipid in HVJ-E: 2.2 pmol/HAU) administration without laser irradiation. The error bar shows a standard deviation. (a) 24 and 48 h incubated after 3 h immersion in HVJ-E or porphyrus envelope (It is the same condition of PDT experiment). (b) 24 and 48 h incubated with HVJ-E or porphyrus envelope.
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ORIGINAL ARTICLES lipid concentrations are shown in Figure 5. The titer of membrane fusion decreased as the concentration of PpIX-lipid increased. It was confirmed that the greater the encapsulated amount of PpIX-lipid was, the less was the membrane fusing capability of HVJ-E.
5. Discussion. 5.1 PDT effect of the porphyrus envelope. The IC50 for PE at 24 h after PDT was 0.04±0.01 µM. The same PDT effect was achieved at a concentration 1/30 of that when PpIX-lipid was administered alone. In the case of no laser irradiation, 3 hour immersion in PE solution produced a significant decrease in the cell survival rate at only 48 h incubation, as shown in Figure 4(b). This means that the main factor of the improvement in PDT efficacy is through the high accumulation of the photosensitizer into the tumor cells due to HVJ-E. Also, since the induction of PpIX-lipid to the cell is mediated through membrane fusion, PpIXlipid which is implanted on the surface of HVJ-E is considered to be re-implanted at the membrane of the cell. It has been reported that photosensitizers located in the cell membrane produce a greater PDT effect than when located in the cytoplasm 17). The localization of PpIX-lipid may have been a factor causing the difference between the PE group and the PpIX-lipid group. The cellular localization of PE and the mechanism that leads to cell death after PDT in more detail will be investigated.
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As shown in Figure 3, the IC50 of PE differed greatly depending on the enclosed amount of PpIXlipid. The reason for this is that less enclosure of PpIXlipid improved the membrane-fusion ability of HVJ-E. In earlier studies, a large amount of PpIX-lipid (44 pmol/HAU) was enclosed in a HVJ-E particle. As shown in Figure 5, an increase in the PpIX-lipid concentration led to a decrease in membrane fusion capability. This study revealed that when PpIX-lipid is enclosed at an amount of 44 pmol/HAU, PE had almost no membrane fusion capability. However, the PE solution used in this study with encapsulated PpIXlipid of 2.2 pmol/HAU, although it may not have been comparable with that of HVJ-E alone, had a membrane fusion titer of approximately 900 HAU/mL. The difference in membrane fusion titer was responsible for the drastic improvement of drug delivery efficiency which led to the improvement of the PDT effect. In actuality, this study revealed that even when the enclosed amount of PpIX-lipid was lowered to 0.35 pmol/HAU, the IC 50 was 6±2 nM, which confirmed that an improved membrane fusion titer leads to improved PDT efficacy. If only the IC50 values were used for comparison, the PpIX induction efficiency of PE with 0.35 pmol/HAU of encapsulated PpIX-lipid was 30 times greater than when PpIX-lipid was administered alone, and was 5250 times greater than the induction of PpIX by 5-ALA administration 12). In this study, laser energy at a wavelength of 405 nm was chosen for its high PpIX absorption and was used with success to achieve highly effective PDT. However, further studies with light sources delivering longer wavelengths are required in the future for in vivo studies where the light penetration depth into living biological tissues becomes a factor, since without absorption, there can be no reaction.
5.2 The anti-tumor effect of the porphyrus envelope.
Fig. 5: The titer of membrane fusion of each porphyrus envelope. The error bar shows a standard deviation.
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The direct effect of HVJ-E for the cancer specific cell death or the anti-tumor effect of HVJ-E and PE is induced by the identification of RNA fragments contained within HVJ-E, by RIG-I. In order to enhance the anti-tumor effect, the fusion of HVJ-E and PE to its targets must take place as much as possible. In this study, the anti-tumor effect of PE solution was inferior to that of HVJ-E in both cases where the tumor cells were immersed in the active solutions for 3 h and 24 h. This was thought to be due to the inferior membrane-fusing ability of PE compared to that of the original HVJ-E. When the enclosed PpIX-lipid was decreased to 0.7
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pmol/HAU, the survival rate of the cells decreased to 65% and was significantly lower (P
