Photosensitizers are critical elements in PDT. Although the quantity and location of PS can, to a certain extent, predict the nature of photodynamic reactions and determine the consequences of the anticancer effect, it is evident that at equivalent cellular levels of PS, there are other factors that might impact the cellular sensitivity as well as phototoxicity. Our cellular models of resistance using closely related porphyrin ethylene glycol derivatives and temoporfin provided new insights into the molecular basis of resistance to PDT.
Resistance to temoporfin is associated with its lysosomal sequestration and changed lysosomal metabolism
Our study confirmed the general consensus that the chemical structure of PS is a key element in the mechanism of resistance to PDT. Temoporfin contains the chlorin core which, unlike the porphyrin central aromatic ring, consists of three pyrroles and one pyrroline coupled through four = CH- linkages. Therefore, temoporfin is not aromatic throughout the entire circumference of the ring24. This structural difference leads to changes in physicochemical properties and in the spectral characteristics25,26. Although the conclusions from existing literature data questioned the possibility of inducing resistance to temoporfin-mediated PDT27, we succeeded in creating resistant cells via multiple selection cycles. The temoporfin-resistant clones differed from porphyrin derivatives in some key characteristics. Specifically, the relocalization of temoporfin to lysosomes (Fig. 5A,B) and more alkaline milieu of the lysosomes as indicated by pH-sensitive probes (Fig. 5B) were observed in temoporfin-resistant cells. We assumed that the lower protonation of temoporfin due to alkalization of lysosomal vesicles resulted in temoporfin aggregation and subsequently inferior fluorescence (Fig. 2A,B) as well as a drop in PDT effectivity (Fig. 2C). This notion was supported by the poor formation of cytotoxic ROS, which are the primary inducers of cell death after light irradiation (Fig. 5C, Supplementary Fig. S4). An alternative explanation based on the increased inactivation of ROS via detoxifying enzymes and activation of heat-shock proteins2 seems to be less likely since no changes in the expression profile of these proteins were found in temoporfin-resistant cells, although they may play a role at the posttranscriptional level. Yet, the molecular mechanism of the change in lysosomal milieu is not clear. A detailed search of microarray data in temoporfin-resistant clones revealed moderately downregulated Atp6ap2, which is a regulator of vacuolar proton ATPase, the protein complex which is responsible, among others, for lysosomal acidity28. This could explain the change of the lysosomal milieu in resistant cells but additional studies are needed to confirm this. The proposed mechanism of lysosomal sequestration does not exclude the possibility of other factors that may have a role in the temoporfin resistance.
Another clue suggesting a role of lysosomal metabolism in temoporfin resistance comes from the study of cross-resistance. Temoporfin-resistant clones retained sensitivity to KP1 and photofrin, whereas they became partially resistant to KP6 (Fig. 2C, orange bars). We believe that this resistance to KP6 is caused by the natural localization of KP6 in lysosomes (Fig. 1B). While lysosomal KP6 in parental cells is able to cause considerable phototoxicity, in temoporfin-resistant clones with changed lysosomal milieu KP6 is less effective. In contrast, KP1, which is localized in ER in both parental and temoporfin-resistant cells, elicits strong phototoxicity (Fig. 2A,C).
Changes in cytoskeleton and cell adhesion
The microarray analysis revealed significant changes in the expression of genes encoding proteins that are associated with the cytoskeleton, cell adhesion (including several components of the integrin pathway), metabolism, and many others in all resistant clones (Supplementary Table S3), which visibly altered cell shape (Fig. 2A) and adhesion behavior. Specifically, temoporfin-resistant clones displayed significant downregulation of a major cytoskeletal protein, vimentin (Supplementary Fig. S3, Supplementary Table S3), and upregulation of γ-tubulin 2. Recent studies have linked the changes in cytoskeleton (actin, α-tubulin) and cellular adhesion mediators (E-cadherin, integrins, fibronectin) to the development of PDT resistance3,29 and metastatic potential of tumor cells30. In animal models, PDT was shown to affect the metastatic potential of various tumors31,32. Interestingly, photosensitization with phthalocyanine Pc4 was linked to vimentin cleavage which precedes poly(ADP-ribose) polymerase (PARP) degradation, a characteristic sign of apoptosis33. It is possible that downregulation of vimentin helps cells to avoid this apoptotic pathway and thus facilitates development of resistance to temoporfin. Temoporfin-resistant clones were also more refractory to trypsinization and showed a high tendency to remain as aggregates. However, the changes in the gene expression which is responsible for these effects and their relevance to the mechanism of resistance remain elusive.
Effect of ABC transporters on KP1 and KP6 resistance
The investigation of KP1 and KP6 resistance showed that the porphyrin structural motif with ethylene glycol substitution on the periphery determines both derivatives as substrates for MDR transport and efflux mediated by ABC transporters. This conclusion, which is in line with the observations made for other PS16, is supported by several lines of evidence. Both KP1 and KP6 PDT-resistant clones were characterized by markedly elevated levels of both Abcb1 transcript (Figs 4 and 6A) and protein (Fig. 6B). Accordingly, resistant cells displayed reduced fluorescence and phototoxicity after KP1 and KP6 exposure (Fig. 2). Moreover, when resistant clones were exposed to the same light dose but increasing doses of KP1 and KP6, the sensitivity to PDT was restored (see Supplementary Fig. S6). This observation demonstrates that high concentrations of PS can saturate the drug pump, which validates the efflux-driven mechanism. The actual doses of PS required for reaching IC80 in KP1-resistant clones were 10–14 times higher than parental cells while only 4–5 times higher in KP6 clones. When these cells were pre-treated with transporter inhibitors (cyclosporin A, probenecid, MK-571), although their specificity was slightly questionable, the fluorescence of KP1 and KP6 as well as the sensitivity toward PDT was, at least in part, increased (Supplementary Fig. S7). Moreover, KP1- and KP6-resistant clones displayed substantial cross-resistance when challenged with KP6 and KP1, respectively (Fig. 2, Supplementary Table S2), hence demonstrating similar mechanisms of resistance.
However, the assessment of cross-resistance indicated a plausible participation of another transporter, ABCA1, whose selective expression was shown by qPCR and Western blot analysis (Fig. 6, Supplementary Table S4). In addition, the possible differential affinity of the transporters to KP1 and KP6 substrates as well as the duration of the selection process might also play a role. For example, KP1 clones expressed a consistently high level of ABCB1 but differed in the levels of ABCA1. Upregulation of ABCA1 was detected in the KP1-1 clone and correlated with the appearance of cross-resistance to KP6, while the KP1-2 clone neither expressed ABCA1 nor was fully resistant to the KP6 challenge (Figs 2 and 6). We hypothesize that cells with upregulated ABCB1 are enriched early during selection due to its high affinity to both KP1 and KP6. Later, with increasing selective pressure (higher light doses), ABCA1 upregulation becomes more important. The establishment of cells resistant to KP1-mediated PDT required fewer additional cycles compared to KP6-resistant cells (Supplementary Table S1), thus isolation of KP1 clones could have occurred at the stage of an increasing but not yet fixed level of ABCA1 transporter in the mixed cell population, which resulted in differences between the clones. In contrast, numerous selection cycles before the isolation of KP6 clones facilitated fixation of ABCA1 expression in the entire population, and thereby expressed in both analyzed clones. Moreover, a potential difference in the affinity of ABCA1 to KP6 and KP1 substrates may be an another reason for the more prominent role of this transporter in KP6 resistance. In this respect, we observed, interestingly, strong upregulation of ABCB1 but not ABCA1 (Fig. 7D) in paclitaxel-resistant cells. These cells were still partially sensitive to KP6-PDT (Fig. 7C), showing that ABCB1 itself was not sufficient to mediate complete drug efflux and cross-resistance (Fig. 7, Supplementary Table S5). Taking into account many other specific factors that affect PDT and paclitaxel resistance, the involvement of ABCA1, particularly in KP6 resistance, is highly probable. To the best of our knowledge, the only published data indicating the elevation of ABCA1 after hypericin-mediated PDT treatment has not sparked much interest34. Interestingly, KP1- and KP6-resistant clones were more sensitive to photofrin-mediated PDT than parental cells (Fig. 2C). Photofrin was recognized as a substrate of the ABCG2 transporter in several reports35,36,37, therefore downregulation of Abcg2 in KP1 and KP6 clones could make these cells more sensitive to photofrin-mediated PDT relatively to parental cells (Fig. 4, Supplementary Table S3). We have provided convincing data via experiments with siRNA-mediated specific knockdown in MCF-7/PacR cells overexpressing ABCB1 that the ABCB1 transporter is responsible for the development of resistance to KP1 and KP6. The cells displayed rescued sensitivity, as revealed by increased fluorescence and relative sensitivity to PDT (Fig. 8). The level of sensitivity differed depending on PS. While reduction of ABCB1 level resulted in the restoration of sensitivity toward KP1, only partial rescue of sensitivity was achieved from the KP6 challenge, suggesting the involvement of ABCA1 in KP6 efflux which was discussed above. To the best of our knowledge, this is the most conclusive demonstration of ABCB1 involvement in PDT resistance induced by porphyrins. In spite of the fact that the overall overexpression of ABCB1 and ABCA1 was identified as the main cause of KP1- and KP6-mediated PDT resistance, the contribution of other factors cannot be ruled out.
The impact of solubility on PS performance and susceptibility to resistance
A major problem with PDT efficacy is generally the low solubility of PS, which consequently leads to aggregation. In some cases, aggregation merely diminishes the activity of the PS, whereas with others it may change its mechanisms of action38. The tetra-ethylene glycol derivatives were originally prepared in our laboratory to improve solubility18,19. The improvement in solubility might be the reason for their higher PDT efficacy in comparison to temoporfin. However, the monomeric form of glycol porphyrins could be more susceptible to efflux by transporters while more lipophilic temoporfin is not effectively pump out.
Our cellular model revealed different molecular mechanisms eliciting PDT resistance to porphyrin derivatives and temoporfin. We found that the resistant cell line variants had distinctive temoporfin- or porphyrin-specific resistance profiles, with nearly non-reciprocal cross-resistance. In temoporfin resistance, the sequestration of PS to lysosomes and their alkalinisation, which results in aggregation, poor ROS formation and low photodynamic efficacy, seems to play a major role. Both KP1 and KP6 porphyrin derivatives, in spite of their different intracellular localization and mechanism of action, become targets of the ABCB1 transporter. The possible involvement of another transporter, ABCA1, is also supported, mainly in the context of KP6 resistance. Furthermore, it was found that PS eliciting resistance by similar mechanisms create cross-resistance, which could be circumvented by drugs that tend to be eliminated differently. Thus, temoporfin resistance can be reverted by using KP1 and photofrin, while KP1 and KP6 resistance can be overcome by photofrin and partly by temoporfin. In addition, several new, along with some previously described, drug resistance-associated genes were identified in each resistant cell line variant. Each PDT resistant cell line variant acquired a unique set of changes that may represent distinct functional subtypes of PDT therapy resistance. Based on these results, we believe that our data contributes to a better understanding of PDT resistance mechanisms, which could in turn lead to more rational design of strategies combining PDT with chemotherapy or other modalities in the treatment of cancer.
18 Feb Mla dissertation xtix
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