Marianne J. Cronjé

Investigating the efficiency of novel metallo-phthalocyanine PDT-induced cell death in MCF-7 breast cancer cells.

BACKGROUND Cancer cells possess an innate resistance to inducers of the death program. Novel phthalocyanines with improved physiochemical properties harbor the potential for use in photodynamic therapy (PDT); a rising treatment alternative preferred for its mostly asymptomatic application and unique mechanism of action. METHODS This study aimed to determine whether in vitro PDT with two new metallo-phthalocyanines (metallo-Pcs), AlPcSmix and GePcSmix, are similarly effective in overcoming resistance to cell death in MCF-7 cells with a brief comparison to an established chemotherapeutic agent, etoposide. Optimum induction of cell death in these cancer cells was initially determined via measurement of cellular respiration and energy production levels. Indications of cytotoxicity and cell stress were evaluated and resultant levels compared to those in cells treated with etoposide. RESULTS Initial findings report AlPcSmix is predominantly more detrimental to cellular function and homeostasis when excited via red light irradiation of 15 J/cm(2). It appears GePcSmix requires higher dosage administrations to achieve similar responses within identical populations. However, due to the mechanism of PDT application, our findings indicate a greater toxic effect with both phthalocyanines when compared to etoposide of higher dosage within MCF-7 cells. CONCLUSION Both phthalocyanines, despite similarity in structure, indicate induction of different cell death response pathways based on their toxicity potential. These therefore show great promise as potential PDT agents for the treatment of cancer.

In vitro changes in mitochondrial potential, aggresome formation and caspase activity by a novel 17‐β‐estradiol analogue in breast adenocarcinoma cells

2‐Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2‐methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2‐ethyl‐3‐O‐sulphamoyl‐estra‐1,3,5(10)16‐tetraene (C19), a non‐commercially available 17‐β‐estradiol analogue, was tested on the breast adenocarcinoma MCF‐7 cell line. The in vitro influence of 24 h exposure to 0.18 μM of C19 on MCF‐7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization‐optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19‐treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19‐treated samples. An increase in the number of cells present in the sub‐G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19‐treated cells. C19 induced both apoptosis and autophagy in MCF‐7 cells. Copyright

The effect of 1:2 Ag(I) thiocyanate complexes in MCF-7 breast cancer cells

There is much interest currently in the design of metal compounds as drugs and various metal compounds are already in clinical use. These include gold(I) compounds such as auranofin and the anti-cancer platinum(II) complex, cisplatin. Bis-chelated gold(I) phosphine complexes have also shown great potential as anticancer agents, however, their efficacy has been limited by their high toxicity. In this study, silver(I) thiocyanate compounds linked to four specific ligands, were synthesized and characterized. These silver-phosphine adducts included [AgSCN{P(4-MeC6H4)3}2]2 (1); [AgSCN{P(4-ClC6H4)3}2]2 (2); [AgSCN{P(4-MeOC6H4)3}2]2 (3); [AgSCN(PPh3)2]2 (4). The compounds were found to be toxic to MCF-7 breast cancer cells while the ligands on their own were not toxic. Our findings further indicate that the silver(I) phosphine compounds induce apoptotic cell death in these breast cancer cells. In addition, the compounds were not toxic to nonmalignant fibroblast cells at the IC50 concentrations. This is an indication that the compounds show selectivity towards the cancer cells.

Novel Porphyrazine Derivatives show Promise for Photodynamic Therapy despite Restrictions in Hydrophilicity

Complexing of ligands to photosensitizers (Ps) has gained popularity by enhancing solubility, cell‐surface recognition and tissue specificity for applications in Photodynamic Therapy (PDT) and fluorescence‐based diagnostics. Here we report on nine carbohydrate‐functionalized porphyrazine (Pz‐galactopyranose/methyl‐ribose) derivatives bearing either H2, Zn(II) or Ni(II) cores for potential use in PDT. Derivatives proved soluble only in organic solvents; dichloromethane (DCM) and tetrahydrofuran (THF). Derivatives were subsequently solubilized using DCM‐based PEG‐DSPE5000‐PBS encapsulation for biological studies due to THF cytotoxicity. Absorption spectra analyses viewed no correlation between core ion, carbohydrate type and peripheral position though encapsulation efficiency (%EE) followed a general order of Zn(II) (60–92%) > H2 (5–34%) > Ni(II) (4–21%). As such, phototoxicity of Zn(II)Pz derivatives were far superior to H2Pz and Ni(II)Pz counterparts following 631.4 nm excitation of MCF‐7 breast cancer cells. Variation was attributed to persistent aggregation and low %EE when regarding the absorption properties recorded. It is therefore believed that revision of the encapsulation method for H2Pz and Ni(II)Pz derivatives would render improved phototoxicity. Zn(II)Pz derivatives show promise as agents for PDT of cancer.

The effects of two metallophthalocyanines on the viability and proliferation of an esophageal cancer cell line.

OBJECTIVE The objective of this study was to establish the influence of two metallophthalocyanine photosensitizers, in their inactive and activated forms, on the cellular reactions of esophageal cancer cells. BACKGROUND DATA Photodynamic therapy (PDT) is an alternative used in the treatment of cancer. During PDT, the activated compound produces cytotoxic singlet molecular oxygen ((1)O(2)), which ultimately leads to cell death. Esophageal cancer has become one of the most common cancers to occur in the world, and the incidence in South Africa is high, especially within the black male population. METHODS Optimal photosensitizer concentration was determined by following the viability of esophageal cancer (SNO) cells treated with a range of concentrations of two metallophthalocyanine photosensitizers, GePcSmix and AlPcSmix, activated by irradiation at a fluence of 20 J/cm(2). Changes in cell morphology were observed after treatment with optimal photosensitizer concentrations, and the effect of the treatment on cell proliferation and cytotoxicity were studied. RESULTS Cell viability decreased in a dose-dependent manner after PDT, while the photosensitizers in their inactive forms did not have an effect on the cells. The altered morphology of cells after PDT was indicative of a necrotic mode of cell death. The optimal photosensitizer concentrations reduced cell proliferation by more than 50% and a significant reduction in cytotoxicity, as detected by lactate dehydrogenase release, was observed following PDT. CONCLUSION Under the studied parameters PDT using GePcSmix and AlPcSmix in vitro could be a useful therapy for esophageal cancer since the photosensitizers alone caused no damage, but cell death is imminent post-PDT.

Apoptotic inducing ability of a novel photosensitizing agent, Ge Sulfophthalocyanine on oesophageal and breast cancer cell lines

Photodynamic therapy (PDT) involves the use of a photosensitizer, which, when activated by light becomes toxic to the cancer cells. Lasers provide light at a specific wavelength required to activate the photosensitizer while the monochromaticity of the lasers at specific wavelengths results in maximum effectiveness of the photosensitizer during treatment. An important property of photosensitizers is that they should absorb light at a long wavelength as the light has to be able to penetrate tissue, and low energy light is able to travel further through tissue than light which absorbs at a shorter wavelength. This study aimed at evaluating the effects of 2 different photosensitizers, Al (AlPcSmix), commercially known as Photosens(R) and Ge (GePcSmix), both from the Phthalocyanine family of sensitizers, on oesophageal (SNO) and breast cancer (MCF-7) cells. Cells were irradiated at 660nm with a power output of 100 mW and a fluence of 10 J/cm2. Cell viability and proliferation were assessed using adenosine triphosphate (ATP) luminescence and alamarBlueTM staining. Lactate dehydrogenase (LDH) activity was used as a measure of cytotoxicity while the Comet assay was used to evaluate DNA damage. Heat shock protein 70 (Hsp70) induction acted as a measure of cellular stress. Both photosensitizers used during the course of this study are effective in targeting malignant cells, and have a cytotoxic effect on these cells when activated using laser irradiation. However, cytotoxic effects were also measured in the absence of laser irradiation, indicating the importance of photosensitizer concentration. Lower concentrations of photosensitizer in the presence of laser irradiation showed greater apoptotic inducing ability than with high concentrations. Morphologically, cells were affected to the detriment despite viability tests indicating the contrary.

The ability of silver(I) thiocyanate 4-methoxyphenyl phosphine to induce apoptotic cell death in esophageal cancer cells is correlated to mitochondrial perturbations

First generation silver(I) phosphines have garnered much interest due to their vast structural diversity and promising anticancer activity. Increasing incidences of cancer, side-effects to chemotherapeutic agents and redevelopment of tumors due to resistance prompts the exploration of alternative compounds showing anticancer activity. This study revealed the effective induction of cell death by a silver(I) thiocyanate 4-methoxyphenyl phosphine complex in a malignant esophageal cell line. Apoptotic cell death was confirmed in treated cells. Moreover, mitochondrial targeting via the intrinsic cell death pathway was evident due to low levels of ATP, altered ROS activity, mitochondrial membrane depolarization, cytochrome c release and caspase-9 cleavage. The complex displayed low cytotoxicity towards two human non-malignant, skin and kidney, cell lines. The findings reported herein give further insight into the selective targeting of silver(I) phosphines and support our belief that this complex shows great promise as an effective chemotherapeutic drug.

Apoptosis-inducing ability of silver(I) cyanide-phosphines useful for anti-cancer studies

Metal-based drugs have shown early promise as anticancer agents suggesting the potential application of silver(I) complexes as apoptosis-inducing agents. The ability of a silver(I) cyanide containing phosphine complex to induce cell death was evaluated in both a malignant (SNO esophageal cancer) and non-malignant (HDF-a skin and HEK293 kidney) cell lines. A dose-dependent decrease in cell viability was observed in the SNO cells. Light microscopy revealed morphological features indicative of apoptotic cell death. The mode of cell death was confirmed as apoptosis by phosphatidylserine externalization, DNA fragmentation and nuclear condensation. Furthermore, both the non-malignant cell lines showed morphological features indicative of apoptosis when exposed to complex 1. We propose the use of this silver(I) cyanide phosphine complex as an highly effective positive apoptosis control for use in anticancer studies of phosphine complexes.

A Comparison of the Toxicity of Mono, Bis, Tris and Tetrakis Phosphino Silver Complexes on SNO Esophageal Cancer Cells

BACKGROUND A broad range of metal-coordinated complexes have been studied for their anticancer activities. However, some of these complexes display high toxicity profiles to non-malignant cells, therefore limiting their use in cancer therapeutics. Aims/Method Several silver(I) triphenylphosphine adducts were prepared as 1:1 to 1:4 ratios of silver nitrate to triphenylphosphine. They were further used to determine their anticancer activity in a malignant SNO esophageal cell line. The silver(I) phosphine adducts include: [Ag(PPh3)]NO3 (1); [Ag(PPh3)2]NO3 (2); [Ag(PPh3)3]NO3 (3) and [Ag(PPh3)4]NO3 (4). In addition, the activity of complexes 1-4 was compared to previously reported complexes [Ag(Ph2P(CH2)2PPh2)2]NO3 (5) and [Ag(Ph2P(CH2)3PPh2)2]NO3 (6). The cytotoxicity of complexes 1-6 was also evaluated in non-malignant human dermal fibroblast cells (HDF-a). RESULTS The majority of the complexes (specifically those containing PPh3) were found to be highly toxic to the SNO cells and less toxic towards HDF-a cells, as determined by the alamarBlue® assay. Morphological studies and flow cytometry confirmed that the silver(I) complexes induced apoptosis in the malignant cells. CONCLUSION These results may have an impact on research related to drug discovery and silver(I) phosphine complexes could be added to the arsenal of anticancer agents in addition to the silver-bis-diphenylphosphinoethane and silver-bis-diphenylphosphinopropane adducts.