Cytotoxic activity of photobleached dyes
To screen photosensitizing dyes for their capacity to generate cytotoxic photoproducts, MC540, 13 structural analogues selected from Günther’s (1) series of second-generation merocyanine dyes, and one selenooxonol dye (Fig. 1) were dissolved in serum-supplemented alpha-medium and photobleached by exposure to cool white fluorescent light. L1210 leukemia cells were subsequently suspended in the photobleached dye solutions and incubated at 37°C in the dark. At the end of the incubation period, cells were washed with dye- and photoproduct-free alpha-medium supplemented with 5% fetal bovine serum and probed for surviving colony-forming cells.
Several combinations of serum concentration, dye concentration, light dose, and incubation time were tried in pilot experiments. All gave qualitatively identical results. A dye concentration of 26 μm, a fetal bovine serum concentration of 12%, a fluence of 97.2 kJ m−2, a cell concentration of 106 mL−1, and an incubation time of 1 h were eventually adopted as standard conditions, (1) because they were similar to those used in preclinical and phase I/II clinical applications of merocyanine-PDT for the extracorporeal purging of autologous hematopoietic stem cell grafts (3–9,23) and (2) because for most cell lines, they generated levels of cytotoxic activity that were within the range of sensitivity of the clonal assay.
As Table 1 shows, all selenobarbituric acid analogues generated photoproducts that were toxic to L1210 leukemia cells. When prepared and tested under standard conditions, photobleached solutions of all selenobarbituric acid analogues reduced the concentration of colony-forming leukemia cells by ≥98%. By contrast, MC540 and its barbituric (MC47) and thiobarbituric (MC1, MC4, MC6, MC7, MC9, MC10, MC17 and MC49) acid analogues generated photoproducts that showed no cytotoxic activity, even if dyes were structurally identical to selone dyes except for the oxygen or sulfur atom at the 2-position of the barbiturate. All selenobarbituric acid analogues (MC54, MC55, MC56 and MC57) generated cytotoxic photoproducts of similar potency regardless of the structure of the aromatic back ring. Thus, an oxidizable selone appeared to be the only structural requirement for cytotoxic activity. The importance of the selone was underscored by experiments with the bis-(1,3-dibutyl selenobarbituric acid) trimethine oxonol dye (16). The oxonol dye lacked the benzene or naphthalene back ring of the merocyanine dyes, but contained two selones instead of a single selone (Fig. 1). In agreement with its higher selone content, the oxonol dye generated cytotoxic photoproducts that had twice the potency of photoproducts generated by equimolar concentrations of selenomerocyanines. Temperature was critical for cytotoxic activity. Very little or no activity was detected when cells were incubated with photoproducts on ice.
Table 1. Structural requirements and protein requirements for generation of cytotoxic and green-fluorescent photoproducts.
|Dye||Descriptor 1||Descriptor 2||Cytotoxic activity||Green fluorescence|
|MC17||Naphth[1,2-d] thiazole||Thiobarbituric||No||Yes (weak)|
| Fetal bovine serum||Yes||Yes|
| Bovine serum albumin||Yes||Yes|
| Human serum albumin||Yes||Yes|
| Carboxymethylated bovine serum albumin||No||Yes|
| Cysteinylated bovine serum albumin||Not done||Yes|
| Human immunoglobulin G (IgG)*||No||No|
| Human low-density lipoprotein||Yes||No|
| Human high-density lipoprotein||Yes||No|
| Albumin-depleted fetal bovine serum||Yes||No|
| HEPES buffer||No||No|
As selone dyes had higher singlet oxygen quantum yields than thione and oxone dyes, selone dyes were photobleached more rapidly than their thione and oxone analogues. This raised the question whether lack of cytotoxic activity was simply the result of inadequate photobleaching. To address this question, light doses were increased four-fold (to 388.8 kJ m−2) for the photobleaching of MC540 but reduced four-fold (to 24.3 kJ m−2) for the photobleaching of MC54. Under these conditions, MC540 was bleached 88%, and MC54 was bleached 85%. Despite virtually identical degrees of photobleaching, only the photoproducts of the selone dye (MC54) showed cytotoxic activity.
Selenomerocyanine-derived cytotoxic photoproducts were active at remarkably low concentrations. When 99%-inhibitory doses were taken as a basis for comparison, MC54-derived photoproducts were about 35–50 times more toxic than selenium dioxide (selenious acid) or sodium selenite and >>50 times more toxic than seleno-DL-methionine or seleno-DL-cystine (Fig. 2).
Figure 2. Cytotoxic activity of photobleached selenomerocyanine dye (MC54) and selected selenium compounds. L1210 leukemia cells were suspended in HEPES-buffered alpha-medium supplemented with 12% fetal bovine serum and MC54-derived photoproducts or Se compounds as indicated, incubated at 37°C for 1 h, washed, and then assayed for surviving in vitro clonogenic cells. Data points reflect mean colony counts of four replicate culture dishes ± standard errors. Most error bars are smaller than the data symbols.
Download figure to PowerPoint
No cytotoxic activity was detected unless selone dyes were photobleached in the presence of serum or certain serum constituents (Table 1). Fetal bovine serum, human serum, bovine serum albumin, human low-density lipoprotein (LDL), and human high-density lipoprotein (HDL) supported the production of cytotoxic activity, whereas human IgG (which did not bind to L1210 target cells) did not.
When MC54 was photobleached in the presence of carboxymethylated bovine albumin (S-carboxymethyl-albumin; less than 0.02 mole sulfhydryl per mole of albumin and no more than 1.5 moles of S-carboxymethyl-cysteine per mole of albumin), no cytotoxic activity was detected within the tested dose range. This suggested that cysteine-34 (CySH-34), the lone thiol of albumin, or a site close to CySH-34 played a role in the generation of cytotoxic activity.
We tried to corroborate this finding with cysteinylated bovine serum albumin (cysteinyl-albumin; less than 0.02 mole sulfhydryl per mole of albumin). However, results were inconclusive because the commercial preparation of cysteinyl-albumin was highly cytotoxic (>95% reduction of clonogenic L1210 cells) in the absence of photoproducts. Additional evidence for a role of CySH-34 in the formation of cytotoxic activity was derived from experiments that exposed tumor cells simultaneously to cytotoxic photoproducts and a noncytotoxic concentration (6 μg mL−1) of cisplatin, a drug known to bind to CySH-34. Cisplatin protected tumor cells from the cytotoxic effects of MC54-derived photoproducts, most likely by competing with cytotoxic photoproducts for the same site.
Serum or an appropriate serum (lipo)protein had to be present during the photobleaching process to support the generation of cytotoxic activity. When selone dyes were first photobleached in ethanol and subsequently mixed with serum- or albumin-containing medium, very little or no cytotoxic activity was detected.
To support the production of cytotoxic activity, serum or serum components did not need to be derived from the same species as the target cells. A comparison of MC54-derived photoproducts generated in the presence of human serum or fetal bovine serum showed that both preparations were equally effective at killing human K562 leukemia cells.
When cytotoxic photoproduct preparations were subjected to ultrafiltration with a molecular weight cut-off of 30 kDa, the cytotoxic activity was retained in the large molecular weight fraction. When proteins in cytotoxic photoproduct preparations were precipitated by the addition of four volumes of cold ethanol, the cytotoxic activity was recovered in the protein precipitate. Thus, in both experiments, the cytotoxic activity behaved like a macromolecule or a conjugate between a small photoproduct and a macromolecule. ICP-MS confirmed that in cytotoxic photoproduct preparations, virtually all Se present was associated with the macromolecular fraction. However, attempts to determine the oxidation state of Se by X-ray photoelectron spectroscopy (a quantitative spectroscopic surface analysis technique that measures the elemental composition of materials and the electronic state of elements) were not successful because the concentration of Se was too low relative to the concentration of protein and buffer salts.
When glutathione, sodium azide, or alpha-tocopherol (1 mm) were present during the photobleaching process, the production of cytotoxic activity was inhibited. Thus, the inhibitory effect of quenchers/scavengers was consistent with (but not strictly diagnostic of) a singlet oxygen-mediated process.
Cytotoxic photoproducts of potency equal to that achieved with the white light source were also obtained with the 574 and 612 nm narrow-band LED light sources, indicating that the photochemical reaction was indeed driven by chromophores that absorbed in the green/orange region of the visible spectrum.
Cytotoxic photoproduct preparations were stable if stored at −80°C. No loss of activity was noticed over a period of 6 months (Fig. 3a). Preparations that were stored at −20°C (Fig. 3b) or 5°C (data not shown) showed a gradual loss of cytotoxic activity. Macroscopic precipitates suggestive of elemental Se were noticed in samples that had lost cytotoxic activity when stored at 5°C for several months. Precipitates were insoluble in water and ethanol. X-ray photoelectron spectroscopy (XPS) of precipitates that had been harvested by centrifugation and washed with water and ethanol confirmed the presence of Se. The oxidation state of Se could not be unequivocally established, as a sodium peak adjacent to the Se 3d peak interfered with the analysis. However, scanning electron microscopy combined with energy dispersive X-ray spectroscopy (a variant of XPS that separately identifies x-rays that are characteristic of each element’s unique atomic structure) clearly showed that the precipitates consisted of Se in oxidation state zero (Fig. 4). Secondary electron images (Fig. 4a) and corresponding EDS spectra taken with the JEOL 840/Kevex instrument showed two distinct regions that were evident throughout the sample, a calcium (Ca)-rich region and sponge-like Se-rich region. A small oxygen (O) signal was consistently associated with the Ca-rich region, but not with the Se-rich region (Fig. 4c,d). Backscatter micrographs taken with the Hitachi S-5000 instrument confirmed that the sponge-like material consisted of a heavier atom (Se) than the crystals that constituted the Ca-rich regions (Fig. 4b). EDS spectra obtained with the Hitachi S-5000/Voyager system confirmed that Se-rich regions contained only trace amounts of O at variable O:Se ratios (data not shown). By contrast, the Ca-rich regions showed strong O and carbon (C) signals at constant O:C:Ca ratios, indicating that the Ca-rich regions consisted of calcium carbonate and the Se-rich region consisted of elemental Se.
Figure 3. Stability of MC54-derived cytotoxic photoproduct-protein conjugates. Material that was stored at −80°C (a) was stable for at least 6 months. Material that was stored at −20°C (b) showed a gradual decline of cytotoxic activity. Fetal bovine serum served as carrier protein, and L1210/L-PAM1 leukemia cells were used to assess cytotoxic potency. Data points reflect mean colony counts of four replicate culture dishes ± standard errors. Most error bars are smaller than the data symbols.
Download figure to PowerPoint
Figure 4. Scanning electron micrographs of crystalline material found after prolonged storage of cytotoxic photoproducts. (a) Secondary electron image obtained with the JEOL 840 instrument. (b) Backscatter image obtained with the Hitachi S-5000 instrument. The sponge-like material backscatters electrons more effectively (brighter image) than the surrounding material, indicating that the sponge-like material was made up of a heavier atom (Se) than the surrounding material. (c and d) EDS spectra of Se-rich and Ca-rich regions obtained with JEOL 840/Kevex instrument. A light gold coating of the sample (to prevent charging effects) explains the gold (Au) signals.
Download figure to PowerPoint
Analysis of photoproduct formation by absorption spectroscopy, fluorescence emission spectroscopy, and mass spectroscopy
All merocyanine dyes were photobleached when exposed to white light in the presence of oxygen. When the selenobarbituric acid analog MC54 (Fig. 1) was dissolved in HEPES buffer (10 mm, pH 7.4) supplemented with fetal bovine serum (12%) and then exposed to white light (27 W m−2) for up to 60 min, the absorption peak of the intact dye (apex of monomer at 625 nm) disappeared rapidly, and a blue-shifted peak with a monomer apex at 598 nm emerged in its place (Fig. 5a). While the intact dye was weakly fluorescent with an emission maximum at 635 nm, the blue-shifted peak of the primary chromophore photoproduct was more intensely fluorescent with an emission maximum at 613 nm (Fig. 5b). These modest blueshifts of the absorption and fluorescence emission spectra were consistent with a minor disruption of the conjugation such as a singlet oxygen-mediated oxidation of the selone and a subsequent substitution of Se by O. The spectral characteristics (peak absorption wavelength, peak fluorescence emission wavelength) of the primary chromophore photoproduct were indeed indistinguishable from the spectral characteristics of dye MC47 (Fig. 1), the authentic barbituric acid analog of MC54 (1,2). The higher fluorescence quantum yield of the primary chromophore photoproduct was also consistent with a replacement of the original selone dye by its oxone analog (1).
Figure 5. Analysis of the photobleaching of selenomerocyanine dye MC54 by absorption spectroscopy (a), fluorescence emission spectroscopy (b) and mass spectroscopy (c–f). Panel (a) shows absorption spectra of native and photobleached MC54 (2.6 μm) dissolved in HEPES buffer (10 mm; pH 7.4) supplemented with 12% fetal bovine serum. Panel (b) shows normalized fluorescence emission spectra of intact MC54, its primary chromophore photoproduct (oxone analog of MC54), and the conjugate of the secondary chromophore photoproduct with albumin. Photobleaching was performed in HEPES buffer supplemented with bovine serum albumin. Panels (c and d) show the mass spectrum of intact MC54: a (M+H) pseudomolecular ion cluster centered on m/z 662.1, a (M+Na) adduct ion cluster centered on m/z 684.1, and a (M-H+2 Na) adduct ion cluster centered on m/z 706.1. The isotope distribution pattern (d) of these clusters was consistent with a Se-containing compound. Panels (e and f) show the mass spectrum of the primary chromophore photoproduct of MC54 generated in 90% ethanol: a (M+H) pseudomolecular ion cluster centered on m/z 598.2, a (M+Na) adduct ion cluster centered on m/z 620.2, and a (MH+2 Na) adduct ion cluster centered on m/z 642.2. The isotope distribution pattern (f) of these clusters was consistent with a compound that did not contain Se, and their masses were consistent with the calculated mass of the oxone analog of MC54.
Download figure to PowerPoint
All selenomerocyanine dyes showed the same initial photobleaching pattern regardless of the structure of their aromatic back rings. They all generated primary chromophore photoproducts that were characterized by moderately blue-shifted absorption and fluorescence emission spectra and enhanced fluorescence quantum yields. While these changes were consistent with a replacement of the original selone dyes by their oxone analogues, an authentic oxone analog for direct comparisons was only available for MC54.
The first step of photobleaching process (replacement of the original selone dye by its oxone analog) also took place in organic solvent (ethanol), provided the solvent contained some water (with dissolved O2) to support the production of singlet oxygen. Mass spectroscopic analyses of photoproducts generated in ethanol showed that the primary chromophore photoproduct of MC54 had a mass consistent with the calculated mass of the dye’s oxone analog (Fig. 5e,f). The mass spectrum of intact MC54 showed the typical isotope distribution of Se (M 74, 0.9%; M 76, 9.0%; M 77, 7.6%; M 78, 23.5%; M 80, 49.8%; M 82, 9.2%; Fig. 5c,d). By contrast, the mass spectrum of primary chromophore photoproduct showed no evidence of Se (Fig. 5f) and matched replacement of Se by O.
When aqueous solutions of MC54 were photoirradiated for prolonged periods of time in the presence of suitable proteins, the primary chromophore photoproduct (oxone analog) was gradually replaced by a secondary chromophore photoproduct that was characterized by a small absorption peak at 499 nm (Fig. 5a) and a strong fluorescence emission peak at 522 nm (Fig. 5b). These major (90–100 nm) blueshifts of the absorption and fluorescence emission spectra were indicative of a major disruption of the conjugation such as an oxidation of the polymethine chain.
As Table 1 shows, the structural requirements for the generation of the green-fluorescent species were different from those for the generation of cytotoxic activity. While only selone dyes were able to generate photoproducts with cytotoxic activity, selone, thione, and oxone dyes were able to generate the green-fluorescent species. While the structure of back ring was irrelevant for the generation of cytotoxic activity, only dyes with S or Se in the donor heterocycle were able to generate the green-fluorescent entity.
Protein requirements were also different (Table 1). At least three serum constituents (albumin, LDL and HDL) supported the generation of cytotoxic activity, whereas only one—albumin—supported the production of the green-fluorescent species (Table 1). Carboxymethylation of albumin interfered with the generation of cytotoxic activity, but neither carboxymethylation nor cysteinylation interfered with the generation of the green-fluorescent photoproduct (Table 1). This suggested that CySH-34 did not play a role in the formation of the green-fluorescent entity.
The green-fluorescent species was retained by ultrafiltration membranes with a cut-off of 30 kDa and was co-precipitated with serum albumin by the addition of four volumes of cold ethanol or acetone. When MC54 was photobleached in the presence of fetal bovine serum and subsequently chromatographed on a Sephadex G-100 gel filtration column, the green-fluorescent material co-eluted with the albumin monomer/dimer peaks. Taken together, these results indicated that the green-fluorescent species was a conjugate between a chromophore photoproduct and serum albumin and that the formation of cytotoxic and green-fluorescent conjugates involved different sites of the albumin molecule.
The formation of green-fluorescent photoproduct-albumin conjugates was a light-dependent process even if the primary chromophore photoproduct (i.e. authentic MC47) was used as a starting material. No green-fluorescent products formed when MC47 was incubated with albumin in the dark. Displacement of oxygen in the buffer by argon, or the addition of sodium azide, alpha-tocopherol, or glutathione (1 mm) inhibited the formation of green-fluorescent photoproduct-albumin conjugates, suggesting that green-fluorescent photoproduct-albumin conjugates were also the product of a singlet oxygen-mediated photochemical process.
The green-fluorescent material was resistant to 5 m guanidine hydrochloride and extraction with chloroform:methanol (2:1). Boiling the material in sodium dodecyl sulfate (2%) and dithiothreitol (100 mm) for 3 min reduced the green-fluorescent peak by 58%.
Unlike cytotoxic conjugates, green-fluorescent photoproduct-albumin conjugates were stable under a wide range of conditions. No loss of fluorescence was noticed when samples were stored at room temperature, 5, −20, or −80°C for up to 6 months. Green-fluorescent conjugates were, however, moderately sensitive to light exposure. This became evident when photobleaching was extended beyond 1 h (97.2 kJ m−2) in an attempt to generate green-fluorescent photoproduct-albumin conjugates that were free of trace amounts of original dye and primary chromophore photoproduct. When white light was used for this purpose, green fluorescence yields peaked after about 4 h of exposure (388.8 kJ m−2) and then progressively decreased to 23% of the peak value after 25 h (2,430 kJ m−2) of exposure. When the 612 nm narrow-band LEDs light source (which did not emit in the 490 nm range) was used for the same purpose, peak fluorescence yields achieved after 4 h of exposure were about 70% higher than those achieved with white light and remained at this level for at least 25 h of continued exposure.
When tumor cells were incubated with photobleached (in the presence of serum or serum albumin) MC54 under conditions that were cytotoxic for target cells, the target cells bound/internalized substantial amounts of green-fluorescent material that were readily detected with the flow cytometer using standard FITC settings. The amount of cell-associated green-fluorescent material was proportional to conjugate concentration and incubation time. Low temperatures, which protected cells against the cytotoxic effect of Se-protein conjugates also inhibited the uptake of green-fluorescent material. Spectroscopic analyses performed on detergent extracts (2% Triton X-100) of conjugate-stained tumor cells confirmed the flow cytometry data.
Fluorescent conjugates prepared with carboxymethylated albumin were bound/internalized by target cells at the same rate as fluorescent conjugates prepared with native albumin. The failure of carboxymethylated albumin to support the formation of cytotoxic conjugates (Table 1) could thus not be explained by a lack of binding/uptake of the carrier protein.
As the formation of fluorescent and cytotoxic photoproduct-protein conjugates was independently regulated, it was possible to generate conjugate preparations that were cytotoxic and fluorescent, cytotoxic but not fluorescent, or fluorescent but not cytotoxic. Side-by-side comparisons of cytotoxic conjugates prepared from MC54 and MC56 (Fig. 1) indicated that at the concentrations used, the presence of green-fluorescent material did neither enhance nor reduce the activity of cytotoxic conjugates. Preparations that were both cytotoxic and fluorescent were used for the majority of experiments because the binding/uptake of green fluorescent photoproduct-albumin conjugates provided a convenient surrogate assay for the binding/uptake of cytotoxic conjugates. On the other hand, MC56-derived preparations that were cytotoxic, but not fluorescent were preferred for experiments that involved the use of green-fluorescent probes such as FITC-labeled antibodies.
Figure 6 summarizes our current understanding of the formation of cytotoxic and green-fluorescent photoproduct-protein conjugates using as a specific example the selone dye, MC54. (1) A portion of the singlet oxygen generated by the photoirradiation of the selone dye oxidizes the selone. (2) The resulting selone oxide is unstable, and the Se atom is substituted by O. Selenium in oxidation state zero and the barbituric acid analog of the original dye are produced as primary photoproducts. (3) If the elemental Se is generated in the presence of serum or suitable serum (lipo)proteins, cytotoxic conjugates are formed. (4) If the chalcogen in the donor heterocycle of the original dye is S or Se, green-fluorescent conjugates are formed with serum albumin by an oxygen-dependent photochemical process.
The stoichiometry of the formation of cytotoxic and green-fluorescent conjugates is not yet fully understood. Experiments with different dye:albumin ratios indicated that dye:albumin molar ratios between 1:1 and 2:1 were saturating for the formation of green-fluorescent photoproduct-albumin conjugates. For the formation of cytotoxic conjugates with albumin, a dye:albumin ratio of about 4.5:1 was saturating. Ratios in excess of 4.5:1 were not explored because of the limited solubility of MC54 in water and concerns about the damaging effects of high concentrations of ethanol on proteins and cells. Taking into consideration the importance of CySH-34 for the formation of cytotoxic conjugates and the fact that the thiol content of commercial preparations of serum albumin is usually ≤0.7 m/m (24), one plausible explanation for a saturating dye:protein ratio of about 4.5:1 is that mercaptalbumin molecules bound 6 or 8 atoms of Se, most likely as cyclic hexamers or octamers—two known stable configurations of elemental Se (25)—whereas nonmercaptalbumin molecules bound no Se.
We tried to use mass spectroscopy to corroborate the proposed stoichiometry of cytotoxic Se-albumin conjugates. However, pilot experiments conducted by the institutional MALDI-TOF facility using cytotoxic conjugates prepared under standard conditions showed only native albumin and no evidence of Se or Se-albumin conjugates. Therefore, we also prepared protein-stabilized elemental Se by an established method (the reduction of selenite by ascorbic acid in the presence of bovine serum albumin) and deliberately used high Se:protein molar ratios of 66:1 and 80:1, respectively, to maximize the chances of detecting Se-albumin conjugates. The product was stable, readily passed through 0.1 μm membrane filters, and had the typical red color of elemental Se, yet the analysis by electrospray ionization spectrometry (performed by M-Scan on a Sciex Q-Star/Pulsar instrument) showed only native albumin and no evidence of Se or Se-albumin conjugates. Thus, it appears that Se-protein conjugates are not amenable to this type of mass spectroscopic analysis.
Cytotoxic Se-protein conjugates as potential anticancer agents
Micromolar concentrations of MC54-derived Se-protein conjugates were cytotoxic to leukemia and lymphoma cells (Fig. 7a,b). An incubation time of 1 h was sufficient to deplete most in vitro clonogenic leukemia cells by ≥5 orders of magnitude, whereas causing little or no damage to normal CD34-positive murine and human bone marrow cells. Normal granulocyte/macrophage progenitors (CFU-GM) showed intermediate sensitivity to cytotoxic conjugates (Fig. 7a,b). As a group, solid tumor cells were less sensitive to cytotoxic conjugates than leukemia and lymphoma cells (Fig. 7c). However, for most solid tumor cell lines, therapeutic indices were still large enough to be of potential therapeutic interest (Fig. 7c,d).
Figure 7. Preferential inactivation of murine leukemia cells (a), human leukemia/lymphoma cells (b), human solid tumor cells (c) and selected drug-resistant mutant tumor cell lines (d–f) by cytotoxic Se-protein conjugates. All tumor cell lines were more sensitive to cytotoxic Se-protein conjugates than normal murine or human CD34-positive bone marrow cells. Most tumor cell lines were also more sensitive than normal murine (mCFU-GM) or human (hCFU-GM) granulocyte/macrophage progenitors (a–c). Melphalan-resistant L1210/L-PAM1 and L1210/L-PAM2 leukemia cells (a) and cisplatin-resistant H69/CDDP small cell lung cancer cells (d) were more sensitive than the corresponding wild-type cells. Adriamycin-resistant P388 leukemia cells (e) were as sensitive as the corresponding wild-type cells. Only one mutant cell line, the adriamycin-resistant HL-60/ADM leukemia (f), was less sensitive than wild-type HL-60 cells. Data points reflect mean colony counts of four replicate culture dishes or mean numbers of CD34-positive bone marrow cells of four determinations ± standard errors. Most error bars are smaller than the data symbols.
Download figure to PowerPoint
Tumor cells died quickly after exposure to selenomerocyanine-derived cytotoxic photoproducts. For example, when L1210 leukemia cells were exposed to MC54-derived photoproducts (26 μm) under standard conditions, 82% of cells were scored as “dead” at the end of the 1 h incubation period based on their staining reaction with propidium iodide (PI). Two hours post incubation, 98% of cells were PI-positive. Similar results were obtained with trypan blue exclusion assays. This rapid onset of cell death was in marked contrast to our experience with MC540-mediated PDT. In MC540-mediated PDT, typically >90% of tumor cells maintain plasma membrane integrity for at least 3 h post treatment, even if clonal assays indicate a ≥4 log depletion of in vitro clonogenic cells (26).
For logistical reasons, the flow cytometric quantitation of CD34-positive cells typically began ≥4 h after the cells’ exposure to cytotoxic conjugates. Absolute cell counts and trypan blue exclusion tests were performed on sample aliquots, while the flow cytometric analysis was in progress. They showed no significant decrease in total cell numbers and no difference in cell viability between treated and untreated samples of normal bone marrow.
Surprisingly, melphalan-resistant mutant L1210/L-PAM1 and L1210/L-PAM2 leukemia cells were more sensitive to cytotoxic conjugates than their wild-type counterparts (Fig. 7a). The higher sensitivity to cytotoxic conjugates was not linked to the mutant cell lines’ drug resistance mechanism (elevated levels of intracellular glutathione), because when mutant cells were grown in medium supplemented with DL-buthionine-[S,R]sulfoximine to inhibit glutathione biosynthesis and restore glutathione levels to wild-type levels, sensitivity to cytotoxic conjugates was not reduced to wild-type levels, but further enhanced. Flow cytometric binding/uptake measurements showed that mutant L1210 cells bound/internalized green-fluorescent photoproduct-albumin conjugates and fluorescein-labeled bovine serum albumin at a higher rate than wild-type cells. This suggested that the enhanced sensitivity to cytotoxic conjugates by mutant cells was the result of enhanced conjugate binding/uptake.
When investigations were extended to four additional drug-resistant mutant tumor cell lines, one mutant cell line (cisplatin-resistant H69/CDDP small cell lung cancer; elevated glutathione-S-transferase-π, metallothionenine and glutathione) was more sensitive (Fig. 7d), and one (adriamycin-resistant HL60/ADR leukemia; MRP-mediated drug efflux) was less sensitive (Fig. 7f) than the corresponding wild-type line. The remaining two mutant cell lines, the adriamycin-resistant P388/ADR leukemia cell line (overexpression of P-glycoprotein; Fig. 7e) and the cisplatin-resistant PC14/CDDP lung adenocarcinoma cell line (reduced drug uptake; data not shown) were as sensitive as the corresponding wild-type cells. The enhanced sensitivity of H69/CDDP cells correlated with enhanced binding/uptake of green-fluorescent photoproduct-albumin conjugate, whereas the reduced sensitivity of HL60/ADR cells correlated with reduced binding/uptake.