Kashif Azizuddin

Bax is essential for mitochondrion-mediated apoptosis but not for cell death caused by photodynamic therapy

The role of Bax in the release of cytochrome c from mitochondria and the induction of apoptosis has been demonstrated in many systems. Using immunocytochemical staining, we observed that photodynamic therapy (PDT) with the photosensitiser Pc 4 induced Bax translocation from the cytosol to mitochondria, and the release of cytochrome c from mitochondria as early signalling for the intrinsic pathway of apoptosis in human breast cancer MCF-7c3 cells. To test the role of Bax in apoptosis, MCF-7c3 cells were treated with Bax antisense oligonucleotides, which resulted in as much as a 50% inhibition of PDT-induced apoptosis. In the second approach, Bax-negative human prostate cancer DU-145 cells were studied. Following PDT, the hallmarks of apoptosis, including the release of cytochrome c from mitochondria, loss of mitochondrial membrane potential, caspase activation, and chromatin condensation and fragmentation, were completely blocked in these cells. Restoration of Bax expression in DU-145 cells restored apoptosis, indicating that the resistance of DU-145 cells to PDT-induced apoptosis is due to the lack of Bax rather than to another defect in the apoptotic machinery. However, despite the inhibition of apoptosis, the Bax-negative DU-145 cells were as photosensitive as Bax-replete MCF-7c3 cells, as determined by clonogenic assay. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to Bax activation.

Association Between the Photodynamic Loss of Bcl-2 and the Sensitivity to Apoptosis Caused by Phthalocyanine Photodynamic Therapy¶

Abstract We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT–induced apoptosis, whether overall cell killing is also sensitized or how upregulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2Δ33-54 and Bcl-2Δ37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2Δ210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2Δ33-54 or Bcl-2Δ37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells.

Structural factors and mechanisms underlying the improved photodynamic cell killing with silicon phthalocyanine photosensitizers directed to lysosomes versus mitochondria.

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4‐loaded cells, membrane components, especially Bcl‐2, are photodamaged and apoptosis, as indicated by activation of caspase‐3 and cleavage of poly(ADP‐ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three‐fold higher affinity constants for liposomes. The hydroxyl‐bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4‐PDT photodamages Bcl‐2 and Bcl‐xL, Pc 181‐PDT causes much less photodamage to Bcl‐2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase‐3‐dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl‐bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway.

Promotion of Photodynamic Therapy-Induced Apoptosis by the Mitochondrial Protein Smac/DIABLO: Dependence on Bax¶

Abstract Photodynamic therapy (PDT) using the second-generation photosensitizer phthalocyanine (Pc) 4 causes mitochondrial damage and induces apoptosis through the release of cytochrome c to the cytosol. Another protein of the mitochondrial intermembrane space, Smac/DIABLO (second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pI), is also released to the cytosol in response to apoptotic stimuli and promotes caspase activation by binding IAP. To investigate the possible role of Smac/DIABLO in apoptosis induced by Pc 4-PDT, we transfected Smac/DIABLO (tagged at its C-terminus with green fluorescent protein [GFP]) into MCF-7c3 cells (human breast cancer MCF-7 cells stably transfected with procaspase-3) and DU-145 cells (human prostate cancer cells that express no Bax because of a frameshift insertion mutation). Confocal microscopy showed that recombinant Smac/DIABLO, like cytochrome c, localized to mitochondria and colocalized with MitoTracker Red. Three hours after exposure of MCF-7c3 cells to PDT (200 nM Pc 4 and 150 mJ/cm2 red light), Smac/DIABLO–GFP, as well as cytochrome c, was found largely in the cytosol. In contrast, for DU-145 cells, both Smac/DIABLO–GFP and cytochrome c remained in the mitochondria after PDT. By staining with Hoechst 33342, typical apoptotic nuclei were observed in MCF-7c3 cells, but not in DU-145 cells, after Pc 4-PDT. These results suggest that the release of Smac/DIABLO from mitochondria may be regulated by a Bax-mediated mechanism and that Smac/DIABLO may cooperate with the cytochrome c–dependent apoptosis pathway. In addition, in MCF-7c3 cells transfected by Smac/DIABLO–GFP, apoptosis induced by Pc 4-PDT was greater than in cells transfected with the GFP vector alone or in untransfected cells, as determined by flow cytometry. Thus, Smac/DIABLO promotes apoptosis after Pc 4-PDT in a Bax-dependent manner and may facilitate the passage of PDT-treated cells through the late steps of apoptosis.

The Death of Human Cancer Cells Following Photodynamic Therapy: Apoptosis Competence is Necessary for Bcl‐2 Protection but not for Induction of Autophagy†

Photodynamic therapy (PDT) is an efficient inducer of apoptosis in many types of cells, except in cells deficient in one or more of the factors that mediate apoptosis. Recent reports have identified autophagy as a potential alternative cell death process following PDT. Here we investigated the occurrence of autophagy after PDT with the photosensitizer Pc 4 in human cancer cells that are deficient in the pro‐apoptotic factor Bax (human prostate cancer DU145 cells) or the apoptosis mediator caspase‐3 (human breast cancer MCF‐7v cells) and in apoptosis‐competent cells (MCF‐7c3 cells that stably overexpress human pro‐caspase‐3 and Chinese hamster ovary CHO 5A100 cells). Further, each of the cell lines was also studied with and without stably overexpressed Bcl‐2. Autophagy was identified by electron microscopic observation of the presence of double‐membrane‐delineated autophagosomal vesicles in the cytosol and by immunoblot observation of the Pc 4‐PDT dose‐ and time‐dependent increase in the level of LC3‐II, a component of the autophagosomal membrane. Autophagy was observed in all of the cell lines studied, whether or not they were capable of typical apoptosis and whether or not they overexpressed Bcl‐2. The presence of stably overexpressed Bcl‐2 in the cells protected against PDT‐induced apoptosis and loss of clonogenicity in apoptosis‐competent cells (MCF‐7c3 and CHO 5A100 cells). In contrast, Bcl‐2 overexpression did not protect against the development of autophagy in any of the cell lines or against loss of clonogenicity in apoptosis‐deficient cells (MCF‐7v and DU145 cells). Furthermore, 3‐methyladenine and wortmannin, inhibitors of autophagy, provided greater protection against loss of viability to apoptosis‐deficient than to apoptosis‐competent cells. The results show that autophagy occurs during cell death following PDT in human cancer cells competent or not for normal apoptosis. Only the apoptosis‐competent cells are protected by Bcl‐2 against cell death.

Caspase-independent apoptosis, in human MCF-7c3 breast cancer cells, following photodynamic therapy, with a novel water-soluble phthalocyanine

A new water-soluble phthalocyanine derivative, 2,3,9,10,16,17,23,24-octakis(3-aminopropyloxy) phthalocyaninato zinc II (PoII) was studied as a photosensitizer for photodynamic therapy (PDT) in MCF-7c3 cells. We report here that PoII and red light induces apoptosis. However, the precise mechanism appears to differ from that induced by PDT with other known phthalocyanines. The present study provides evidence that in the case of PoII, caspases do not participate in the apoptotic response. PoII-PDT-treated cells exhibited chromatin condensation and phosphatidylserine (PS) externalization. In the absence of light activation, PoII had no detectable cytotoxic effect. An early event upon PoII-PDT was photodamage to lysosomes, suggesting that they are the primary sites of action. Moreover, the treatment induces Bid activation, mitochondrial swelling and translocation of apoptosis-inducing factor (AIF) to the nucleus. An atypical proteolysis of poly(ADP-ribose) polymerase (PARP) indicative of calpain-like activation was observed. These data support the notion that an alternative mechanism of caspase-independent apoptosis was found in PoII-photosensitized cells.

Targeting of mitochondria by 10-N-alkyl acridine orange analogues: Role of alkyl chain length in determining cellular uptake and localization

10-N-Nonyl acridine orange (NAO) is used as a mitochondrial probe because of its high affinity for cardiolipin (CL). Targeting of NAO may also depend on mitochondrial membrane potential. As the nonyl group has been considered essential for targeting, a systematic study of alkyl chain length was undertaken; three analogues (10-methyl-, 10-hexyl-, and 10-hexadecyl-acridine orange) were synthesized and their properties studied in phospholipid monolayers and breast cancer cells. The shortest and longest alkyl chains reduced targeting, whereas the hexyl group was superior to the nonyl group, allowing very clear and specific targeting to mitochondria at concentrations of 20-100 nM, where no evidence of toxicity was apparent. Additional studies in wild-type and cardiolipin-deficient yeast cells suggested that cellular binding was not absolutely dependent upon cardiolipin.

The Peripheral Benzodiazepine Receptor in Photodynamic Therapy with the Phthalocyanine Photosensitizer Pc 4

Abstract The peripheral benzodiazepine receptor (PBR) is an 18 kDa protein of the outer mitochondrial membrane that interacts with the voltage-dependent anion channel and may participate in formation of the permeability transition pore. The physiological role of PBR is reflected in the high-affinity binding of endogenous ligands that are metabolites of both cholesterol and heme. Certain porphyrin precursors of heme can be photosensitizers for photodynamic therapy (PDT), which depends on visible light activation of porphyrin-related macrocycles. Because the apparent binding affinity of a series of porphyrin analogs for PBR paralleled their ability to photoinactivate cells, PBR has been proposed as the molecular target for porphyrin-derived photocytotoxicity. The phthalocyanine (Pc) photosensitizer Pc 4 accumulates in mitochondria and structurally resembles porphyrins. Therefore, we tested the relevance of PBR binding on Pc 4–PDT. Binding affinity was measured by competition with 3H-PK11195, a high-affinity ligand of PBR, for binding to rat kidney mitochondria (RKM) or intact Chinese hamster ovary (CHO) cells. To assess the binding of the Pc directly, we synthesized 14C-labeled Pc 4 and found that whereas Pc 4 was a competitive inhibitor of 3H-PK11195 binding to the PBR, PK11195 did not inhibit the binding of 14C–Pc 4 to RKM. Further, 14C–Pc 4 binding to RKM showed no evidence of saturation up to 10 μM. Finally, when Pc 4–loaded CHO cells were exposed to activating red light, apoptosis was induced; Pc 4–PDT was less effective in causing apoptosis in a companion cell line overexpressing the antiapoptotic protein Bcl-2. For both cell lines, PK11195 inhibited PDT-induced apoptosis; however, the inhibition was transient and did not extend to overall cell death, as determined by clonogenic assay. The results demonstrate (1) the presence of low-affinity binding sites for Pc 4 on PBR; (2) the presence of multiple binding sites for Pc 4 in RKM and CHO cells other than those that influence PK11195 binding; and (3) the ability of high supersaturating levels of PK11195 to transiently inhibit apoptosis initiated by Pc 4–PDT, with less influence on overall cell killing. We conclude that the binding of Pc 4 to PBR is less relevant to the photocytotoxicity of Pc 4–PDT than are other mitochondrial events, such as photodamage to Bcl-2 and that the observed inhibition of Pc 4–PDT–induced apoptosis by PK11195 likely occurs through a mechanism independent of PBR.

Photodynamic therapy-induced death of HCT 116 cells: Apoptosis with or without Bax expression

Cell death following photodynamic therapy (PDT) with the photosensitizer Pc 4 involves the intrinsic pathway of apoptosis. To evaluate the importance of Bax in apoptosis after PDT, we compared the PDT responses of Bax-proficient (Bax+/−) and Bax knock-out (BaxKO) HCT116 human colon cancer cells. PDT induced a slow apoptotic process in HCT Bax+/− cells following a long delay in the activation of Bax and release of cytochrome c from mitochondria. Although cytochrome c was not released from mitochondria following PDT in BaxKO cells, an alternative mechanism of caspase-dependent apoptosis with extensive chromatin and DNA degradation was found in these cells. This alternative process was less efficient and slower than the normal apoptotic process observed in Bax+/− cells. Early events upon PDT, such as the loss of mitochondrial membrane potential, photodamage to Bcl-2, and activation of p38 MAP kinase, were observed in both HCT116 cell lines. In spite of differences in the efficiency and mode of apoptosis induced by PDT in the Bax+/− and BaxKO cells, they were found to be equally sensitive to killing by PDT, as determined by loss of clonogenicity. Thus, for Pc 4-PDT, the commitment to cell death occurs prior to and independent of Bax activation, but the process of cellular disassembly differs in Bax-expressing vs. non-expressing cells.

Apoptosis mechanisms related to the increased sensitivity of Jurkat T-cells vs A431 epidermoid cells to photodynamic therapy with the phthalocyanine Pc 4.

To examine the clinical applicability of Pc 4, a promising second‐generation photosensitizer, for the photodynamic treatment of lymphocyte‐mediated skin diseases, we studied the A431 and Jurkat cell lines, commonly used as surrogates for human keratinocyte‐derived carcinomas and lymphocytes, respectively. As revealed by ethyl acetate extraction and absorption spectrophotometry, uptake of Pc 4 into the two cell lines was linear with Pc 4 concentration and similar on a per cell basis but greater in Jurkat cells on a per mass basis. Flow cytometry showed that uptake was linear at low doses; variations in the dose–response for uptake measured by fluorescence supported differential aggregation of Pc 4 in the two cell types. As detected by confocal microscopy, Pc 4 localized to mitochondria and endoplasmic reticulum in both cell lines. Jurkat cells were much more sensitive to the lethal effects of phthalocyanine photodynamic therapy (Pc 4‐PDT) than were A431 cells, as measured by a tetrazolium dye reduction assay, and more readily underwent morphological apoptosis. In a search for molecular factors to explain the greater photosensitivity of Jurkat cells, the fate of important Bcl‐2 family members was monitored. Jurkat cells were more sensitive to the induction of immediate photodamage to Bcl‐2, but the difference was insufficient to account fully for their greater sensitivity. The antiapoptotic protein Mcl‐1 was extensively cleaved in a dose‐ and caspase‐dependent manner in Jurkat, but not in A431, cells exposed to Pc 4‐PDT. Thus, the greater killing by Pc 4‐PDT in Jurkat compared with A431 cells correlated with greater Bcl‐2 photodamage and more strongly to the more extensive Mcl‐1 degradation. Pc 4‐PDT may offer therapeutic advantages in targeting inflammatory cells over normal keratinocytes in the treatment of T‐cell‐mediated skin diseases, such as cutaneous lymphomas, dermatitis, lichenoid tissue reactions and psoriasis, and it will be instructive to evaluate the role of Bcl‐2 family proteins, especially Mcl‐1, in the therapeutic response.