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Catalytic therapy of cancer with porphyrins and ascorbate
Cancer Letters 252 (2007) 216–224 www.elsevier.com/locate/canlet
Catalytic therapy of cancer with porphyrins and ascorbate Nadejda Rozanova (Torshina) a
a,*
, Jin Z. Zhang a, Diane E. Heck
b
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA b Department of Toxicology, Rutgers University, Piscataway, NJ 08854, USA
Received 28 September 2006; received in revised form 19 December 2006; accepted 20 December 2006
Abstract Catalytic therapy (CT) is a cancer treatment modality based on the generation of reactive oxygen species (ROS) using a combination of substrate molecules and a catalyst. The most frequently used substrate and catalyst pair is ascorbate/Co phthalocyanine (PcCo). In the present study, porphyrins containing transition metal ions as catalysts in place of PcCo were studied. Porphyrins that are expected to be as efficient as phthalocyanines, but may have fewer side effects, were analyzed. ROS production through the combined use of ascorbate and porphyrins decreased the number of breast cancer tumor cells by 20–40% after a single in vitro treatment, as compared to control cells. Treatment with ascorbate in conjunction with porphyrins stimulated apoptosis and disrupted the cell cycle. These treatments enhanced apoptosis by 20–40% when compared to treatments with ascorbate and porphyrins. In addition, the number of cells accumulating in the sub G0/G1 stage of the cell cycle increased from 3- to 10-fold, potentially reflecting that the treatment was highly effective in inducing DNA damage in the tumor cells, suggesting that porphyrins may be beneficial as a CT catalyst in the treatment of cancer. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Therapy; Cancer; Catalytic; Ascorbate; Porphyrins; Cell cycle; Apoptosis; DNA; Sub G0/G1
1. Introduction Catalytic therapy (CT) is a cancer treatment modality that employs a transition metal complex as a catalyst and a second molecule as a substrate. Catalytic therapy, which is similar to photodynamic therapy (PDT), is an approach to cancer treatment. This radiation-based approach for the treatment of solid malignancies involves the systemic or local administration of a photosensitizing agent followed *
Corresponding author. Tel.: +1 8314291625. E-mail address: [email protected] (N. Rozanova (Torshina)).
by irradiation with appropriate wavelength visible light. In the presence of molecular oxygen, photodynamic treatment results in extensive tumor cell death, necrosis, apoptosis, and significant damage to tumor vasculature. Photodynamic therapy has proved to be successful in the treatment of a broad range of diverse solid tumors; however, its use is limited to tissues and areas accessible to light or light-producing devices [1–3]. In contrast, CT is potentially a more versatile cancer treatment modality, which, although also based on the generation of reactive oxygen species (ROS), uses a combination of substrate molecules and a catalyst in place of light irradiation [4]. Most often the combination
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.12.026
N. Rozanova (Torshina) et al. / Cancer Letters 252 (2007) 216–224
of ascorbate, as a substrate, and phthalocyanine dyes containing transition metal ions, as catalysts, is used. Mechanisms underlying the antitumor action of CT are similar to X-ray therapy and PDT cancer treatments, in that CT’s actions are dependent on the production of ROS, which subsequently induces oxidative degradation of critical cellular molecules and organelles [5–8]. Compared to traditional chemotherapy and PDT, CT has the potential to become a preferred treatment for an array of diverse malignancies. It has been found that a combination of cobalt or iron phthalocyanine and sodium ascorbate has high antitumor activity. This system is highly effective, with a success rate similar to that of PDT. The effectiveness of CT has been demonstrated in in vitro experiments with porphyrin-like moieties (vitamin B12) and its derivatives, as catalysts, and with ascorbate as a substrate [9–15]. Several in vivo experiments with the same CT systems have also been reported [16–28]. A combination of teraphthal (cobalt (II) octa-4,5-carboxyphthalocyanine) and ascorbate has been approved for Phase II clinical trials in Russia [29]. Ascorbate is currently considered the most suitable substrate for CT. It has been well recognized that some human tumors accumulate ascorbate more than normal tissues do [30,31]. It has also been demonstrated that ascorbate may be a pro-drug for the formation of H2O2 in tumor cells [32]. In addition, formation of H2O2 and OH by photosensitizers such as methylene blue, hematoporphyrin, and texaphyrins in the presence of ascorbate and without light has been observed [33–40]. While PDT is limited to regions of the body accessible to light illumination, CT does not require light, thereby making it more flexible and adaptable for treatment of poorly accessible neoplasms. In cancer treatment, CT functions by various mechanisms including direct cell killing, damage to the tumor vasculature, and stimulation of inflammation and nonspecific or specific immune effector cells [41]. It has been suggested that both phthalocyanine and porphyrin complexes are highly promising for
OH
OH
O O
O O
O2
HO
+ H2O2
HO
Pc-Co HO
OH
O
O
Fig. 1. Catalytic oxygenation of ascorbate by Pc Co.
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catalytic therapy [42]. The process of catalytic oxygenation of ascorbate by PcCo is shown in Fig. 1. The mechanism of H2O2 generation has been studied previously [43]. The present studies have been initiated to determine the possibility of using porphyrins to replace PcCo in CT. Here, we show that this combination is indeed active: CT with porphyrins led to the suppression of tumor growth up to 40%. Cell death and damage are exhibited by increases in apoptosis and cell cycle disruption. In addition, porphyrins are expected to have fewer side effects than phthalocyanine. Our confidence is based on our extensive previous work in the area of phthalocyanine toxicity [44–49]. Porphyrins occur widely in nature and they play very important roles in various biological processes. Their toxicity is very low, as has been shown [50–52]. 2. Materials and methods 2.1. Cell culture and chemicals MCF-7 human breast cancer cells and B-16 mouse melanoma cells and normal human keratinocytes were obtained from ATCC (Manassas, VA) and maintained in 8% CO2 at 37 C in RPMI 1640 (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen, Carlsbad, CA). Standard chemicals were purchased from Sigma (St. Louis, MO). For cell growth assays, cells were plated in six-well plates (10,000 cells per well); treatment with CT was initiated a day after cell seeding at concentrations indicated in the text. To determine the amount of apoptosis and cell cycle distribution, cells also were plated in six-well plates (10,000 cells per well); treatment with CT was initiated 4 days after cell seeding (before cells achieved full confluence) at concentrations indicated in the text. Hematoporphyrin IX base and cobalt (III) protoporphyrin IX chloride were obtained from Frontier Scientific Inc. (Logan, Utah). 2.2. Statistical analysis All experiments were repeated at least three times in the course of this study. Three control groups were used: (a) untreated cells, (b) cells treated by activation mixture, and (c) cells treated by porphyrins without activation mixture. Student t-test calculations were used. Representative results are presented. Plus or minus standard deviation (±SD) was calculated where appropriate. ANalysis Of VAriance between groups (ANOVA) method has been used as well.
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2.3. Ground-state absorption spectra and static emission spectra Ground-state absorption spectra were recorded on an HP-8452A UV–vis spectrophotometer with 2 nm resolution. A mixture of hematoporphyrin 0.05 mM, ascorbate 0.01 mM, CuCl2 0.02 mM, and IgG 1 mg/ml in PBS (phosphate-buffered saline) was used. Static emission spectra were measured with a Perkin-Elmer LS-50B fluorometer. 2.4. Cell growth suppression Twenty-four hours prior to CT treatment, 0.5 ml of MCF-7 cells in Dulbecco’s modified Eagle’s medium (DMEM) was seeded in six-well plates. Sample size involved one well with three to six parallel measurements. Porphyrin solutions in distilled water of 0.005 mM were added to each well of the six-well plates and incubated for 1 h. We used three types of control cells: cells treated by solvents (untreated control); cells treated by activation mixture; cells treated by porphyrins. After 1 h, DMEM was exchanged for fresh DMEM without porphyrins and the catalytic mixture (ascorbate 0.07 mM and CuSO4 0.07 mM) was added to the wells. Cells were incubated with the catalytic mixture for 1 h, then DMEM was changed to growth DMEM, and the cells were allowed to grow at 37 C in a CO2 incubator. Cells were measured 6 days after treatment, using a Coulter Counter Model Z B1, Coulter Electronic Inc., Hialeah, FL. Viability analysis revealed no significant levels of cell death in untreated cells. 2.5. Cell cycle analysis Four days prior to CT treatment, cells were seeded in six-well plates. CT treatment was carried out as described above. Control and treated cells were harvested, washed twice in PBS, fixed with 70% ethanol for 30 min at room temperature, washed three times in PBS, resuspended in the staining buffer (10 mM Pipes, pH 6.8, 0.1 M NaCl, 0.1% Triton X-100, and 2 mM MgCl2), treated with DNase-free RNase I (100 lg/ml) for 30 min at room temperature, and stained with propidium iodide (PI, Molecular
Probes, Eugene, OR) at a concentration of 10 lg/ml. Stained cells were analyzed by flow cytometry using a Beckman Coulter flow cytometer. 2.6. Annexin V and PI staining Four days prior to CT treatment, cells were seeded in six-well plates. CT treatment was carried out as described above. Both adherent and detached cells from untreated and CT-exposed samples were collected, washed twice in PBS, and double-labeled with Annexin V and PI according to the manufacturer’s protocol (Sigma, St Louis, MO). Control populations consisted of unstained cells and cells stained with only Annexin V or PI. Cells were analyzed by flow cytometry using a Beckman Coulter flow cytometer. The fluorescence emission of 10,000 cells/treatment was collected in each channel. 3. Results and discussion 3.1. Ground-state absorption spectra and static emission spectra In initial studies, CT with ascorbate and Cu2+ ions led to changes in the porphyrin absorbance, in particular in the full-width half-maximum (FWHM) of the Soret and the Q region bands of the UV–vis porphyrin spectra (Table 1). The observed spectral changes indicated that bleaching of porphyrins was associated with ROS production. It was observed that the addition of protein to the porphyrin mixture reduced the bleaching of porphyrins in CT with the activation mixture. As can be seen from Table 1, the changes enhanced alterations after CT in the Soret, and more Q band regions were found in samples without proteins than in the presence of proteins. Previous studies from our laboratory indicate that porphyrins and phthalocyanines form complexes with proteins via p–p interactions between the aromatic ligands of the protein and the tetrapyrrolic macrocycle of the porphyrin ring and that, in the presence of proteins, ROS causes more degradation to proteins than porphyrins [53–56]. Potentially, this effect underlies our current observation that degradation of the porphyrins was diminished in the presence of proteins.
Table 1 Ground-state absorption spectra of hematoporphyrin (Photofrin) Mixtures
Soret FWHM (nm), k 389 nm
A, k 500 nm
A, k 530 nm
A, k 560 nm
HP HP HP HP
72 35 70 65
0.12 – 0.12 –
0.05 0.23 0.05 0.11
0.04 0.34 0.05 0.15
in in in in
PBS PBS + AM PBS + protein PBS + protein + AM
Q (1, 0)
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Fluorescence
20
1
15
2
10
3
5 0 560
580
600 620 Wavelengh (nm)
640
600 620 Wavelength (nm)
640
Fluorescence
20 15 10
1 2 3
5 0 560
580
Fig. 2. (a) Static emission spectra of hematoporphyrin : 1 in PBS, 2 in PBS + CuCl2, 3 in PBS + CuCl2 + ascorbate using 426 nm excitation. (b) Static emission spectra of protoporphyrin IX base: 1 in PBS with protein, 2 in PBS with protein + CuCl2, 3 in PBS with protein + CuCl2 + ascorbate.
Static emission spectra show degradation of the porphyrins after CT as well (Fig. 2). It has been found that static emission spectra decrease in the following order (Fig. 2a): hematoporphyrin in PBS > hematoporphyrin in PBS + CuCl2 > hematoporphyrin in PBS + CuCl2 + ascorbate. Additions of Cu2+ ions led to changes in the porphyrin emission. The observed spectral changes indicated connection between porphyrin and Cu2+ ions. The same order was seen in the presence of protein (Fig. 2b), but static emission spectra of protoporphyrin in PBS with protein + CuCl2 show a higher intensity than without protein. This could be due to interaction of Cu2+ with protein. Meanwhile, static emission spectra of protoporphyrin in PBS with protein + CuCl2 + ascorbate show similar intensity with and without protein. Together, these results indicate that self-degradation of porphyrin during CT treatments is minimalized by the presence of protein. 3.2. Effects of CT on cell growth We next determined the effects of CT on cell growth (Fig. 3). Cells treated with the activation system alone (without porphyrins) were used as a control. In these experiments the formula X = [(O · 100%)/C] 100% was used to calculate cell growth. Here O is the number of cells treated by CT, C is the number of cells treated by the activation system alone (control cells), and X is the percent decrease in the number of cells after treatment with CT in comparison with cells treated by the activation system alone. At the same time, the number of normal keratinocytes that have been used as normal
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cells control did not show any significant decrease (Fig. 3). Therefore, as has been stated in Section 1, CT could not have damaged normal cells as they do not accumulate a sufficient concentration of CT components. However, this has not been explored in detail in the current studies. We found that CT treatment with porphyrins dramatically inhibited breast cancer cell proliferation. The combined use of ascorbate and porphyrins resulted in a decrease in the number of cancer cells by 20–40% after a single in vitro treatment. In addition, growth inhibition was dependent on the dose of CT. At maximal doses (0.05 mM of porphyrin), no tumor cells survived (Figs. 4 and 5). However, at these high concentrations the individual components could have some direct effects on cell death as well; this has not been explored in detail in the current studies (Fig. 4). 3.3. Cellular DNA analysis The cellular response to the stress induced by treatment with anticancer agents is a key determinant of drug activity. A pivotal role in this response is played by checkpoint proteins that control the normal passage of cells through the cell cycle. There is evidence that cancer cells often have defects in one checkpoint control, which makes them more vulnerable to inhibition of a second checkpoint, thereby enhancing the overall response to treatment [57]. Therefore, we examined next the effects of CT treatment on the cell cycle distribution of MCF-7 human breast cancer cells. In these experiments, the influence of porphyrins and activation mixture on the cell cycle was examined using propidium iodide (PI) in conjunction with flow cytometry. Cell DNA content reflecting the G0/G1, S, G2/M stages of the cell cycle as well as sub G0/G1 cells (apoptotic cells) was measured. As demonstrated in Tables 2 and 3, treatment with CT and porphyrins significantly increased the percentage of apoptotic cells (sub G0/G1 cells), the percentage of cells arrested at the G2/ M check point, and the number of dead MCF-7 breast cancer cells. These findings prompted us to further evaluate the apoptotic effects of CT treatment. In these studies the early stages of apoptosis of cancer cells after CT treatment were evaluated by flow cytometry using Annexin 5, a fluorescent indicator dye which binds to phosphatidylserine in the cell membrane, and the viable die propidium iodide. Changes in the plasma membrane of the cell surface are one of the earliest features of cells undergoing apoptosis. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35–36 kDa Ca2+-dependent phospholipid-binding protein with a high affinity for PS; binding to cells with exposed PS is indicative of the early stages of apoptosis. We have also used another vital stain, propidium iodide, which is
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N. Rozanova (Torshina) et al. / Cancer Letters 252 (2007) 216–224 0 1
2
3
4
5
6
4
5
6
-5
Cell growth, (%)
-10 -15 -20 -25 -30 -35 -40 -45 Kind of CT 0 1
2
3
-5
Cell growth, (%)
-10 -15 -20 -25 -30 -35 -40 -45 -50 Kind of CT
Fig. 3. (a) Decrease in MCF-7 breast cancer cells and normal keratinocytes growth following CT treatment (P < 0.05). 1, control (cells treated by AM); 2, CoP; 3, CoP + AM; 4, HP; 5, HP + AM; 6, normal keratinocytes with activation mixture. (b) Repetition of experiments. CoP, Co protoporphyrin (0.005 mM); HP, hematoporphyrin (0.005 mM); AM, activation (ascorbic acid 0.07 mM and CuSO4 0.07 mM).
excluded from viable cells in our analysis. As demonstrated in Tables 2 and 3, the percentages of dead cells increased to 6-fold in cells treated with CoP, HP, and activation mixture when compared to cells treated by the activation mixture alone. It is clear that CT with porphyrins significantly increased both the number of cells undergoing apoptosis and the percentage of dead MCF-7 breast cancer cells. Together, the results of these studies indicate that both CoP and HP CT treatments result in extensive death of tumor cells, and that both these treatments are effective in activating apoptosis. Furthermore, our findings are consistent with the induction of a block in the G2M stage of the cell cycle following HP treatment. An important issue with CT as well as with all other drug therapies is the potential for discrepancies between the results of in vitro and in vivo studies. Drug efficacy can be decreased in vivo due to metabolism of the drug or interactions with other biological structures, or due to small accumulation of components in tumor cells. Thus, there it is necessary to transport drugs (e.g., catalysts,
and the activation mixture) to tumors safely without changing, destroying, or decreasing the concentration, and to achieve a high concentration of components in tumor cells. We have solved this problem in one of our previous studies using magnetically guided liposomes loaded with photosensitizer in in vivo PDT experiments [58]. It has been shown that this guided transport dramatically increased the concentration of photosensitizer at the tumor site and PDT efficiency. This method has been recently rediscovered by University at Buffalo researchers. And it also has been shown in their finding that drug delivery concept in which an applied magnetic field directs the accumulation in tumor cells of customdesigned may lead to treatments that exploit the advantages of PDT and that have the potential to reduce drug accumulation in normal tissues [59]. Increased effectiveness of CT may result from the use of the same guided delivery system of components. Moreover, new products such as polymeric- and ceramic-based nanoparticles, combined with specific antibodies, may be tested
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Fig. 4. B-16 mouse melanoma cells after big doses CT treatment: (a) with Co porphyrin 0.05 mM and activation mixture ascorbic acid 0.175 mM and CuSO4 0.175 mM; (b) with Co porphyrin 0.05 mM; (c) with activation mixture -ascorbic acid 0.175 mM and CuSO4 0.175 mM; (d) ascorbic acid 0.175 mM.
HP
Table 2 Parameters changed following CT with CoP on MCF-7 cancer cells
CoP (repetition)
Parameters
CoP
CoP + AM
AM
Sub G0/G1 (%) P(s); P(A) G2/M (%) P(s); P(A) Dead cells (%) P(s); P(A) Annexin-peak shift (units) P(s); P(A)
5.7 ± 2.3 0.014; 0.0001 15.0 ± 1.4 0.01; 0.0006 10.3 ± 0.8 0.000; 0.0001 1.2 ± 0.2
18.0 ± 4.6
0.8 ± 0.3 0.003 9.5 ± 1.4 0.001 9.9 ± 0.9 0.001 0.5 ± 0.06
140 CoP 120
HP (repetition)
100
CoP-C Hp-C
80 60 40 20
0.009; 0.0001
8.7 ± 1.9 63.2 ± 1.6 2.1 ± 0.3
0.001
P(s), Student’s test; P(A), ANOVA test. In ANOVA method, we compared the results of (1) control, (2) AM treated, (3) porphyrins treated, and (4) porphyrins + AM-treated cells.
0 0
0.01
0.02
0.03
0.04
0.05
0.06
Porphyrins concentrations, (mM)
Fig. 5. The dose dependence curves for CT of MCF-7 cells. CoP, Co porphyrin with activation mixture (ascorbate 0.07 mM and CuSO4 0.07 mM); HP, hematoporphyrin with activation mixture (ascorbate 0.07 mM and CuSO4 0.07 mM); repetition of CoP with activation mixture; repetition of HP with activation mixture; CoP-C, control (without activation mixture); Hp-C, control (without activation mixture).
as well. This complete system of guided transport (magnetically guided, antibody combined drug-loaded nanoparticles) may help to deliver the components of CT to tumors with little change or loss. This could significantly speed up in vivo CT research and development, and this will be investigated in our future research.
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Table 3 Parameters changed following CT with HP on MCF-7 cancer cells Parameters
HP
HP + AM
AM
Sub G0/G1 (%) P(s); P(A) G2/M (%) P(s); P(A) Dead cells (%) P(s); P(A) Annexin-peak shift (units) P(s); P(A)
1.5 ± 0.8 0.000; 0.001 12.6 ± 2.4 0.008; 0.0001 33.5 ± 13.5 0.026; 0.0001 0.6 ± 0.02
18.4 ± 1.4
0.8 ± 0.3 0.001 9.5 ± 1.4 0.009 9.9 ± 0.9 0.0001 0.5 ± 0.06
0.000; 0.0001
20.3 ± 1.3 62.1 ± 4.8 1.9 ± 0.1
0.0001
P(s), Student’s test; P(A), ANOVA test. In ANOVA method, we compared the results of (1) control, (2) AM-treated, (3) porphyrins treated, and (4) porphyrins + AM-treated cells.
4. Conclusion The current study found that CT based on porphyrin results in ROS production, thereby increasing the percentage of apoptotic cells and decreasing the growth of MCF-7 breast cancer cells. We conclude that CT treatment with a porphyrin catalyst leads to the damage of cancer cells and suppression of tumor growth. CT using a porphyrin catalyst fulfilled important quality criteria, including adequate control, randomization, baseline comparability, outcome measures, adequate statistical analysis, and reproducibility. Reproducibility was met by conducting two separate experiments under identical conditions, using three biological indicators of cell proliferation and viability. Further research using a larger number of measured parameters, cell lines, as well as in vivo experiments for a better understanding of the tumoricidal mechanisms of CT using porphyrins as catalysts is needed. Acknowledgments We thank Dr. Anna Vetrano and Dr. Michael Hamblin for their useful discussion of the results, and Dr. Arlene Haffa for critical reading of the manuscript. This work was supported by CA93798 and CA100994. References [1] M. Triesscheijn, P. Baas, J.H. Schellens, F.A. Stewart, Photodynamic therapy in oncology, Oncologist 9 (2006) 1034–1044. [2] S.B. Brown, E.A. Brown, I. Walker, The present and future role of photodynamic therapy in cancer treatment, Lancet Oncol. 5 (2004) 497–508.
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Catalytic therapy of cancer with porphyrins and ascorbate Nadejda Rozanova (Torshina) a
a,*
, Jin Z. Zhang a, Diane E. Heck
b
Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA b Department of Toxicology, Rutgers University, Piscataway, NJ 08854, USA
Received 28 September 2006; received in revised form 19 December 2006; accepted 20 December 2006
Abstract Catalytic therapy (CT) is a cancer treatment modality based on the generation of reactive oxygen species (ROS) using a combination of substrate molecules and a catalyst. The most frequently used substrate and catalyst pair is ascorbate/Co phthalocyanine (PcCo). In the present study, porphyrins containing transition metal ions as catalysts in place of PcCo were studied. Porphyrins that are expected to be as efficient as phthalocyanines, but may have fewer side effects, were analyzed. ROS production through the combined use of ascorbate and porphyrins decreased the number of breast cancer tumor cells by 20–40% after a single in vitro treatment, as compared to control cells. Treatment with ascorbate in conjunction with porphyrins stimulated apoptosis and disrupted the cell cycle. These treatments enhanced apoptosis by 20–40% when compared to treatments with ascorbate and porphyrins. In addition, the number of cells accumulating in the sub G0/G1 stage of the cell cycle increased from 3- to 10-fold, potentially reflecting that the treatment was highly effective in inducing DNA damage in the tumor cells, suggesting that porphyrins may be beneficial as a CT catalyst in the treatment of cancer. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Therapy; Cancer; Catalytic; Ascorbate; Porphyrins; Cell cycle; Apoptosis; DNA; Sub G0/G1
1. Introduction Catalytic therapy (CT) is a cancer treatment modality that employs a transition metal complex as a catalyst and a second molecule as a substrate. Catalytic therapy, which is similar to photodynamic therapy (PDT), is an approach to cancer treatment. This radiation-based approach for the treatment of solid malignancies involves the systemic or local administration of a photosensitizing agent followed *
Corresponding author. Tel.: +1 8314291625. E-mail address: [email protected] (N. Rozanova (Torshina)).
by irradiation with appropriate wavelength visible light. In the presence of molecular oxygen, photodynamic treatment results in extensive tumor cell death, necrosis, apoptosis, and significant damage to tumor vasculature. Photodynamic therapy has proved to be successful in the treatment of a broad range of diverse solid tumors; however, its use is limited to tissues and areas accessible to light or light-producing devices [1–3]. In contrast, CT is potentially a more versatile cancer treatment modality, which, although also based on the generation of reactive oxygen species (ROS), uses a combination of substrate molecules and a catalyst in place of light irradiation [4]. Most often the combination
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.12.026
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of ascorbate, as a substrate, and phthalocyanine dyes containing transition metal ions, as catalysts, is used. Mechanisms underlying the antitumor action of CT are similar to X-ray therapy and PDT cancer treatments, in that CT’s actions are dependent on the production of ROS, which subsequently induces oxidative degradation of critical cellular molecules and organelles [5–8]. Compared to traditional chemotherapy and PDT, CT has the potential to become a preferred treatment for an array of diverse malignancies. It has been found that a combination of cobalt or iron phthalocyanine and sodium ascorbate has high antitumor activity. This system is highly effective, with a success rate similar to that of PDT. The effectiveness of CT has been demonstrated in in vitro experiments with porphyrin-like moieties (vitamin B12) and its derivatives, as catalysts, and with ascorbate as a substrate [9–15]. Several in vivo experiments with the same CT systems have also been reported [16–28]. A combination of teraphthal (cobalt (II) octa-4,5-carboxyphthalocyanine) and ascorbate has been approved for Phase II clinical trials in Russia [29]. Ascorbate is currently considered the most suitable substrate for CT. It has been well recognized that some human tumors accumulate ascorbate more than normal tissues do [30,31]. It has also been demonstrated that ascorbate may be a pro-drug for the formation of H2O2 in tumor cells [32]. In addition, formation of H2O2 and OH by photosensitizers such as methylene blue, hematoporphyrin, and texaphyrins in the presence of ascorbate and without light has been observed [33–40]. While PDT is limited to regions of the body accessible to light illumination, CT does not require light, thereby making it more flexible and adaptable for treatment of poorly accessible neoplasms. In cancer treatment, CT functions by various mechanisms including direct cell killing, damage to the tumor vasculature, and stimulation of inflammation and nonspecific or specific immune effector cells [41]. It has been suggested that both phthalocyanine and porphyrin complexes are highly promising for
OH
OH
O O
O O
O2
HO
+ H2O2
HO
Pc-Co HO
OH
O
O
Fig. 1. Catalytic oxygenation of ascorbate by Pc Co.
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catalytic therapy [42]. The process of catalytic oxygenation of ascorbate by PcCo is shown in Fig. 1. The mechanism of H2O2 generation has been studied previously [43]. The present studies have been initiated to determine the possibility of using porphyrins to replace PcCo in CT. Here, we show that this combination is indeed active: CT with porphyrins led to the suppression of tumor growth up to 40%. Cell death and damage are exhibited by increases in apoptosis and cell cycle disruption. In addition, porphyrins are expected to have fewer side effects than phthalocyanine. Our confidence is based on our extensive previous work in the area of phthalocyanine toxicity [44–49]. Porphyrins occur widely in nature and they play very important roles in various biological processes. Their toxicity is very low, as has been shown [50–52]. 2. Materials and methods 2.1. Cell culture and chemicals MCF-7 human breast cancer cells and B-16 mouse melanoma cells and normal human keratinocytes were obtained from ATCC (Manassas, VA) and maintained in 8% CO2 at 37 C in RPMI 1640 (Invitrogen, Carlsbad, CA), supplemented with 10% heat-inactivated FCS (HyClone, Logan, UT), 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin (Invitrogen, Carlsbad, CA). Standard chemicals were purchased from Sigma (St. Louis, MO). For cell growth assays, cells were plated in six-well plates (10,000 cells per well); treatment with CT was initiated a day after cell seeding at concentrations indicated in the text. To determine the amount of apoptosis and cell cycle distribution, cells also were plated in six-well plates (10,000 cells per well); treatment with CT was initiated 4 days after cell seeding (before cells achieved full confluence) at concentrations indicated in the text. Hematoporphyrin IX base and cobalt (III) protoporphyrin IX chloride were obtained from Frontier Scientific Inc. (Logan, Utah). 2.2. Statistical analysis All experiments were repeated at least three times in the course of this study. Three control groups were used: (a) untreated cells, (b) cells treated by activation mixture, and (c) cells treated by porphyrins without activation mixture. Student t-test calculations were used. Representative results are presented. Plus or minus standard deviation (±SD) was calculated where appropriate. ANalysis Of VAriance between groups (ANOVA) method has been used as well.
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2.3. Ground-state absorption spectra and static emission spectra Ground-state absorption spectra were recorded on an HP-8452A UV–vis spectrophotometer with 2 nm resolution. A mixture of hematoporphyrin 0.05 mM, ascorbate 0.01 mM, CuCl2 0.02 mM, and IgG 1 mg/ml in PBS (phosphate-buffered saline) was used. Static emission spectra were measured with a Perkin-Elmer LS-50B fluorometer. 2.4. Cell growth suppression Twenty-four hours prior to CT treatment, 0.5 ml of MCF-7 cells in Dulbecco’s modified Eagle’s medium (DMEM) was seeded in six-well plates. Sample size involved one well with three to six parallel measurements. Porphyrin solutions in distilled water of 0.005 mM were added to each well of the six-well plates and incubated for 1 h. We used three types of control cells: cells treated by solvents (untreated control); cells treated by activation mixture; cells treated by porphyrins. After 1 h, DMEM was exchanged for fresh DMEM without porphyrins and the catalytic mixture (ascorbate 0.07 mM and CuSO4 0.07 mM) was added to the wells. Cells were incubated with the catalytic mixture for 1 h, then DMEM was changed to growth DMEM, and the cells were allowed to grow at 37 C in a CO2 incubator. Cells were measured 6 days after treatment, using a Coulter Counter Model Z B1, Coulter Electronic Inc., Hialeah, FL. Viability analysis revealed no significant levels of cell death in untreated cells. 2.5. Cell cycle analysis Four days prior to CT treatment, cells were seeded in six-well plates. CT treatment was carried out as described above. Control and treated cells were harvested, washed twice in PBS, fixed with 70% ethanol for 30 min at room temperature, washed three times in PBS, resuspended in the staining buffer (10 mM Pipes, pH 6.8, 0.1 M NaCl, 0.1% Triton X-100, and 2 mM MgCl2), treated with DNase-free RNase I (100 lg/ml) for 30 min at room temperature, and stained with propidium iodide (PI, Molecular
Probes, Eugene, OR) at a concentration of 10 lg/ml. Stained cells were analyzed by flow cytometry using a Beckman Coulter flow cytometer. 2.6. Annexin V and PI staining Four days prior to CT treatment, cells were seeded in six-well plates. CT treatment was carried out as described above. Both adherent and detached cells from untreated and CT-exposed samples were collected, washed twice in PBS, and double-labeled with Annexin V and PI according to the manufacturer’s protocol (Sigma, St Louis, MO). Control populations consisted of unstained cells and cells stained with only Annexin V or PI. Cells were analyzed by flow cytometry using a Beckman Coulter flow cytometer. The fluorescence emission of 10,000 cells/treatment was collected in each channel. 3. Results and discussion 3.1. Ground-state absorption spectra and static emission spectra In initial studies, CT with ascorbate and Cu2+ ions led to changes in the porphyrin absorbance, in particular in the full-width half-maximum (FWHM) of the Soret and the Q region bands of the UV–vis porphyrin spectra (Table 1). The observed spectral changes indicated that bleaching of porphyrins was associated with ROS production. It was observed that the addition of protein to the porphyrin mixture reduced the bleaching of porphyrins in CT with the activation mixture. As can be seen from Table 1, the changes enhanced alterations after CT in the Soret, and more Q band regions were found in samples without proteins than in the presence of proteins. Previous studies from our laboratory indicate that porphyrins and phthalocyanines form complexes with proteins via p–p interactions between the aromatic ligands of the protein and the tetrapyrrolic macrocycle of the porphyrin ring and that, in the presence of proteins, ROS causes more degradation to proteins than porphyrins [53–56]. Potentially, this effect underlies our current observation that degradation of the porphyrins was diminished in the presence of proteins.
Table 1 Ground-state absorption spectra of hematoporphyrin (Photofrin) Mixtures
Soret FWHM (nm), k 389 nm
A, k 500 nm
A, k 530 nm
A, k 560 nm
HP HP HP HP
72 35 70 65
0.12 – 0.12 –
0.05 0.23 0.05 0.11
0.04 0.34 0.05 0.15
in in in in
PBS PBS + AM PBS + protein PBS + protein + AM
Q (1, 0)
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Fluorescence
20
1
15
2
10
3
5 0 560
580
600 620 Wavelengh (nm)
640
600 620 Wavelength (nm)
640
Fluorescence
20 15 10
1 2 3
5 0 560
580
Fig. 2. (a) Static emission spectra of hematoporphyrin : 1 in PBS, 2 in PBS + CuCl2, 3 in PBS + CuCl2 + ascorbate using 426 nm excitation. (b) Static emission spectra of protoporphyrin IX base: 1 in PBS with protein, 2 in PBS with protein + CuCl2, 3 in PBS with protein + CuCl2 + ascorbate.
Static emission spectra show degradation of the porphyrins after CT as well (Fig. 2). It has been found that static emission spectra decrease in the following order (Fig. 2a): hematoporphyrin in PBS > hematoporphyrin in PBS + CuCl2 > hematoporphyrin in PBS + CuCl2 + ascorbate. Additions of Cu2+ ions led to changes in the porphyrin emission. The observed spectral changes indicated connection between porphyrin and Cu2+ ions. The same order was seen in the presence of protein (Fig. 2b), but static emission spectra of protoporphyrin in PBS with protein + CuCl2 show a higher intensity than without protein. This could be due to interaction of Cu2+ with protein. Meanwhile, static emission spectra of protoporphyrin in PBS with protein + CuCl2 + ascorbate show similar intensity with and without protein. Together, these results indicate that self-degradation of porphyrin during CT treatments is minimalized by the presence of protein. 3.2. Effects of CT on cell growth We next determined the effects of CT on cell growth (Fig. 3). Cells treated with the activation system alone (without porphyrins) were used as a control. In these experiments the formula X = [(O · 100%)/C] 100% was used to calculate cell growth. Here O is the number of cells treated by CT, C is the number of cells treated by the activation system alone (control cells), and X is the percent decrease in the number of cells after treatment with CT in comparison with cells treated by the activation system alone. At the same time, the number of normal keratinocytes that have been used as normal
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cells control did not show any significant decrease (Fig. 3). Therefore, as has been stated in Section 1, CT could not have damaged normal cells as they do not accumulate a sufficient concentration of CT components. However, this has not been explored in detail in the current studies. We found that CT treatment with porphyrins dramatically inhibited breast cancer cell proliferation. The combined use of ascorbate and porphyrins resulted in a decrease in the number of cancer cells by 20–40% after a single in vitro treatment. In addition, growth inhibition was dependent on the dose of CT. At maximal doses (0.05 mM of porphyrin), no tumor cells survived (Figs. 4 and 5). However, at these high concentrations the individual components could have some direct effects on cell death as well; this has not been explored in detail in the current studies (Fig. 4). 3.3. Cellular DNA analysis The cellular response to the stress induced by treatment with anticancer agents is a key determinant of drug activity. A pivotal role in this response is played by checkpoint proteins that control the normal passage of cells through the cell cycle. There is evidence that cancer cells often have defects in one checkpoint control, which makes them more vulnerable to inhibition of a second checkpoint, thereby enhancing the overall response to treatment [57]. Therefore, we examined next the effects of CT treatment on the cell cycle distribution of MCF-7 human breast cancer cells. In these experiments, the influence of porphyrins and activation mixture on the cell cycle was examined using propidium iodide (PI) in conjunction with flow cytometry. Cell DNA content reflecting the G0/G1, S, G2/M stages of the cell cycle as well as sub G0/G1 cells (apoptotic cells) was measured. As demonstrated in Tables 2 and 3, treatment with CT and porphyrins significantly increased the percentage of apoptotic cells (sub G0/G1 cells), the percentage of cells arrested at the G2/ M check point, and the number of dead MCF-7 breast cancer cells. These findings prompted us to further evaluate the apoptotic effects of CT treatment. In these studies the early stages of apoptosis of cancer cells after CT treatment were evaluated by flow cytometry using Annexin 5, a fluorescent indicator dye which binds to phosphatidylserine in the cell membrane, and the viable die propidium iodide. Changes in the plasma membrane of the cell surface are one of the earliest features of cells undergoing apoptosis. In apoptotic cells, the membrane phospholipid phosphatidylserine (PS) is translated from the inner to the outer leaflet of the plasma membrane, thereby exposing PS to the external cellular environment. Annexin V is a 35–36 kDa Ca2+-dependent phospholipid-binding protein with a high affinity for PS; binding to cells with exposed PS is indicative of the early stages of apoptosis. We have also used another vital stain, propidium iodide, which is
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2
3
4
5
6
4
5
6
-5
Cell growth, (%)
-10 -15 -20 -25 -30 -35 -40 -45 Kind of CT 0 1
2
3
-5
Cell growth, (%)
-10 -15 -20 -25 -30 -35 -40 -45 -50 Kind of CT
Fig. 3. (a) Decrease in MCF-7 breast cancer cells and normal keratinocytes growth following CT treatment (P < 0.05). 1, control (cells treated by AM); 2, CoP; 3, CoP + AM; 4, HP; 5, HP + AM; 6, normal keratinocytes with activation mixture. (b) Repetition of experiments. CoP, Co protoporphyrin (0.005 mM); HP, hematoporphyrin (0.005 mM); AM, activation (ascorbic acid 0.07 mM and CuSO4 0.07 mM).
excluded from viable cells in our analysis. As demonstrated in Tables 2 and 3, the percentages of dead cells increased to 6-fold in cells treated with CoP, HP, and activation mixture when compared to cells treated by the activation mixture alone. It is clear that CT with porphyrins significantly increased both the number of cells undergoing apoptosis and the percentage of dead MCF-7 breast cancer cells. Together, the results of these studies indicate that both CoP and HP CT treatments result in extensive death of tumor cells, and that both these treatments are effective in activating apoptosis. Furthermore, our findings are consistent with the induction of a block in the G2M stage of the cell cycle following HP treatment. An important issue with CT as well as with all other drug therapies is the potential for discrepancies between the results of in vitro and in vivo studies. Drug efficacy can be decreased in vivo due to metabolism of the drug or interactions with other biological structures, or due to small accumulation of components in tumor cells. Thus, there it is necessary to transport drugs (e.g., catalysts,
and the activation mixture) to tumors safely without changing, destroying, or decreasing the concentration, and to achieve a high concentration of components in tumor cells. We have solved this problem in one of our previous studies using magnetically guided liposomes loaded with photosensitizer in in vivo PDT experiments [58]. It has been shown that this guided transport dramatically increased the concentration of photosensitizer at the tumor site and PDT efficiency. This method has been recently rediscovered by University at Buffalo researchers. And it also has been shown in their finding that drug delivery concept in which an applied magnetic field directs the accumulation in tumor cells of customdesigned may lead to treatments that exploit the advantages of PDT and that have the potential to reduce drug accumulation in normal tissues [59]. Increased effectiveness of CT may result from the use of the same guided delivery system of components. Moreover, new products such as polymeric- and ceramic-based nanoparticles, combined with specific antibodies, may be tested
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Fig. 4. B-16 mouse melanoma cells after big doses CT treatment: (a) with Co porphyrin 0.05 mM and activation mixture ascorbic acid 0.175 mM and CuSO4 0.175 mM; (b) with Co porphyrin 0.05 mM; (c) with activation mixture -ascorbic acid 0.175 mM and CuSO4 0.175 mM; (d) ascorbic acid 0.175 mM.
HP
Table 2 Parameters changed following CT with CoP on MCF-7 cancer cells
CoP (repetition)
Parameters
CoP
CoP + AM
AM
Sub G0/G1 (%) P(s); P(A) G2/M (%) P(s); P(A) Dead cells (%) P(s); P(A) Annexin-peak shift (units) P(s); P(A)
5.7 ± 2.3 0.014; 0.0001 15.0 ± 1.4 0.01; 0.0006 10.3 ± 0.8 0.000; 0.0001 1.2 ± 0.2
18.0 ± 4.6
0.8 ± 0.3 0.003 9.5 ± 1.4 0.001 9.9 ± 0.9 0.001 0.5 ± 0.06
140 CoP 120
HP (repetition)
100
CoP-C Hp-C
80 60 40 20
0.009; 0.0001
8.7 ± 1.9 63.2 ± 1.6 2.1 ± 0.3
0.001
P(s), Student’s test; P(A), ANOVA test. In ANOVA method, we compared the results of (1) control, (2) AM treated, (3) porphyrins treated, and (4) porphyrins + AM-treated cells.
0 0
0.01
0.02
0.03
0.04
0.05
0.06
Porphyrins concentrations, (mM)
Fig. 5. The dose dependence curves for CT of MCF-7 cells. CoP, Co porphyrin with activation mixture (ascorbate 0.07 mM and CuSO4 0.07 mM); HP, hematoporphyrin with activation mixture (ascorbate 0.07 mM and CuSO4 0.07 mM); repetition of CoP with activation mixture; repetition of HP with activation mixture; CoP-C, control (without activation mixture); Hp-C, control (without activation mixture).
as well. This complete system of guided transport (magnetically guided, antibody combined drug-loaded nanoparticles) may help to deliver the components of CT to tumors with little change or loss. This could significantly speed up in vivo CT research and development, and this will be investigated in our future research.
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Table 3 Parameters changed following CT with HP on MCF-7 cancer cells Parameters
HP
HP + AM
AM
Sub G0/G1 (%) P(s); P(A) G2/M (%) P(s); P(A) Dead cells (%) P(s); P(A) Annexin-peak shift (units) P(s); P(A)
1.5 ± 0.8 0.000; 0.001 12.6 ± 2.4 0.008; 0.0001 33.5 ± 13.5 0.026; 0.0001 0.6 ± 0.02
18.4 ± 1.4
0.8 ± 0.3 0.001 9.5 ± 1.4 0.009 9.9 ± 0.9 0.0001 0.5 ± 0.06
0.000; 0.0001
20.3 ± 1.3 62.1 ± 4.8 1.9 ± 0.1
0.0001
P(s), Student’s test; P(A), ANOVA test. In ANOVA method, we compared the results of (1) control, (2) AM-treated, (3) porphyrins treated, and (4) porphyrins + AM-treated cells.
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