Since the introduction of Auranofin in 1985 there has been no new clinically approved gold containing drugs introduced. Although promising results were achieved with a gold(I) phosphine complex [Au(dppe) 2]Cl (Hoke et al., 1991; Mckeage et al., 2002), this compound was never entered into clinical trials due to its toxicity to normal tissue such as the liver and heart (Smith et al.,1989). Six novel derivatives of [Au(dppe) 2]Cl were developed and synthesized to identify possible new candidates with improved tumour specificity compared to [Au(dppe) 2]Cl and cisplatin. Human cervical carcinoma cells (HeLa) were used for an initial toxicity screening. IC50’s obtained for [Au(dppe) 2]Cl and cisplatin were 0.661 and 0.710 µM respectively. Three mixed novel derivatives (MM4, MM5 and MM6) displayed IC50’s ranging between 0.026 and 0.103 µM. These compounds were then selected to be tested further for selectivity and cytotoxicity on various malignant and normal cell lines. MM4 showed selectivity for ovarian, prostate, cervical and breast cancer cells, while MM5 was the most effective against ovarian, colon, prostate, cervical and breast cancer cells. MM6 was most active against ovarian, colon, prostate, cervical and breast cancer cells. The experimental compounds had much higher IC50’s when tested on the normal cells, which indicates selectivity for cancer cells. The octanol/water partition coefficient (lipophilicity) of all the experimental compounds was measured to determine the lipophilicity of the compounds. [Au(dppe) 2]Cl was found to be strongly lipophilic; while the novel compounds had varying degrees of lipo- and hydrophilicity. The octanol/water partition coefficient (lipophilicity) was also used to establish whether there is a correlation between the lipophilicity, IC50 and tumour specificity. In this study no correlation was found between these parameters. [Au(dppe) 2]Cl is known to have an effect on the mitochondrial membrane potential of cells. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on mitochondrial membrane potential. PHA stimulated human lymphocytes and a human undifferentiated leukemia T-cell line (Jurkat cells) were used in these experiments. [Au(dppe) 2]Cl, MM4, MM5 and MM6 depolarized the mitochondrial membranes of PHA stimulated lymphocytes significantly, while only [Au(dppe) 2]Cl depolarized the mitochondrial membranes of the Jurkat cells significantly, indicating that a different mechanism of action might be operational. MM4, MM5, MM6 and cisplatin were compared to [Au(dppe) 2]Cl for effects on plasma membrane potential. PHA stimulated human lymphocytes and Jurkat cells were used in these experiments. [Au(dppe) 2]Cl and MM6 depolarized the plasma membranes of both PHA stimulated lymphocytes and the Jurkat cells significantly. In order to determine whether the depolarization of mitochondrial and plasma membranes was a precursor for apoptosis, experiments were done to determine whether MM4, MM5 and MM6 induce apoptosis in Jurkat cells. [Au(dppe) 2]Cl and cisplatin were added for comparison. [Au(dppe) 2]Cl, cisplatin, MM4 and MM6 did induce apoptosis in Jurkat cells, but MM5 did not. The effect of [Au(dppe) 2]Cl, cisplatin, MM4, MM5 and MM6 on the cell cycle of Jurkat cells was determined to establish whether the experimental compounds altered this process. [Au(dppe) 2]Cl, MM4, MM5 and MM6 arrested the cell cycle in the G1 phase and cisplatin did so in the S phase. In order to determine whether the inhibition of cell growth and partition coefficient of the experimental compounds is related to the uptake of the drug, radio labeled drug uptake experiments were carried out with 198Au labeled [Au(dppe) 2]Cl, MM5 and MM6. Two different types of ovarian cancer cells were used for these studies. One cell line was sensitive to cisplatin (A2780) and the other was resistant to cisplatin (A2780 cis). Results obtained from these experiments showed that the uptake of these experimental compounds was dependent on their octanol/water partition coefficient. However, the inhibition of cell growth did not correlate with the uptake of these compounds by the cells that were tested. To confirm the octanol/water partition coefficient and drug uptake results, 198Au labelled [Au(dppe) 2]Cl, MM5 and MM6 were testedin vivo for bio distribution in rats. [Au(dppe) 2]Cl (lipophilic) had higher bio distribution compared to MM5 and MM6 (hydrophilic). Conclusion The experimental compounds show low IC50’s combined with increased tumour specificity. This indicates that these compounds have great potential to target tumour cells selectively and should be investigated further as anti-cancer agents. / Dissertation (MSc)--University of Pretoria, 2008. / Pharmacology / unrestricted

A ação das metaloproteinases de matriz (MMP-2, MMP-9 e MT1-MMP) é necessária para degradação da membrana basal em carcinomas da cérvice uterina. O objetivo deste trabalho consistiu na avaliação da expressão das metaloproteinases MMP -2, -9 e MT1-MMP, do gene supressor de metástase RECK e do inibidor tecidual de MMPs (TIMP-2) em modelo de células de neoplasia da cérvice-uterina cultivadas em substratos de matriz extracelular. As linhagens celulares de carcinoma de cérvice uterina SiHa, CaSki, ambas HPV 16 positivas, e C33A, HPV negativa, foram cultivadas em gel de colágeno tipo I, Matrigel e plástico. Avaliou-se o crescimento, invasão, expressão gênica, através de ensaios de real-time PCR, e atividade de metaloproteinases, através de ensaios de zimografia. Os resultados demonstraram que estas linhagens de carcinoma cervical quando cultivadas em gel de colágeno tipo I apresentaram uma diminuição no crescimento, morfologia modificada na presença de substrato de matriz extracelular, e que nas linhagens HPV positivas há um aumento da expressão de MMP-2, MT1-MMP e TIMP-2 e da atividade de pró-MMP-2 em relação à linhagem HPV negativa. Observou-se também que, RECK apresentou maior expressão gênica em CaSki associada à atividade de pró-MMP-2. MMP-9 apresentou muito baixa expressão gênica em todas as linhagens e condições estudadas. Quando analisamos as linhagens separadamente, observamos que o Matrigel influenciou a expressão gênica de MMP-2, e que o gel de colágeno tipo I aparece como indutor da atividade de pró-MMP-2 em todas as linhagens. Em conclusão, nossos resultados mostram que a expressão de MMP-2, MT1-MMP e TIMP-2 e que a atividade de pró-MMP-2 estão aumentadas nas células HPV positivas, sugerindo que o HPV está associado com a expressão de MMPs e TIMP-2. / Matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in three human invasive cervical carcinoma cell lines. Two HPV-16- positive cell lines (SiHa and CaSki), HPV-negative cell line (C33A) were cultured either onto type-I collagen gel, Matrigel or plastic, in order to recreate their three-dimensional growth environment and evaluate the growth and invasion of the cells and expression of these genes using quantitative real-time PCR (Q-PCR). We also analyzed the gelatinolytic activity of MMP-2 and -9 by zymography. We found that the growth curves carcinoma cells are decreased and cells morphology are modified in ECM substrate. HPV-positive cell lines expressed higher levels of MMP-2, MT1-MMP and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with pro-MMP-2 activity. The Matrigel substrate influence MMP-2 expression for SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP -2, MT1-MMP and TIMP-2 and pro-MMP-2 activity in HPV-positive cells than in HPV-negative cells.

A ação das metaloproteinases de matriz (MMP-2, MMP-9 e MT1-MMP) é necessária para degradação da membrana basal em carcinomas da cérvice uterina. O objetivo deste trabalho consistiu na avaliação da expressão das metaloproteinases MMP -2, -9 e MT1-MMP, do gene supressor de metástase RECK e do inibidor tecidual de MMPs (TIMP-2) em modelo de células de neoplasia da cérvice-uterina cultivadas em substratos de matriz extracelular. As linhagens celulares de carcinoma de cérvice uterina SiHa, CaSki, ambas HPV 16 positivas, e C33A, HPV negativa, foram cultivadas em gel de colágeno tipo I, Matrigel e plástico. Avaliou-se o crescimento, invasão, expressão gênica, através de ensaios de real-time PCR, e atividade de metaloproteinases, através de ensaios de zimografia. Os resultados demonstraram que estas linhagens de carcinoma cervical quando cultivadas em gel de colágeno tipo I apresentaram uma diminuição no crescimento, morfologia modificada na presença de substrato de matriz extracelular, e que nas linhagens HPV positivas há um aumento da expressão de MMP-2, MT1-MMP e TIMP-2 e da atividade de pró-MMP-2 em relação à linhagem HPV negativa. Observou-se também que, RECK apresentou maior expressão gênica em CaSki associada à atividade de pró-MMP-2. MMP-9 apresentou muito baixa expressão gênica em todas as linhagens e condições estudadas. Quando analisamos as linhagens separadamente, observamos que o Matrigel influenciou a expressão gênica de MMP-2, e que o gel de colágeno tipo I aparece como indutor da atividade de pró-MMP-2 em todas as linhagens. Em conclusão, nossos resultados mostram que a expressão de MMP-2, MT1-MMP e TIMP-2 e que a atividade de pró-MMP-2 estão aumentadas nas células HPV positivas, sugerindo que o HPV está associado com a expressão de MMPs e TIMP-2. / Matrix metalloproteinases (MMPs) -2, -9, and MT1-MMP are required for basement membrane degradation in cervical carcinoma. We evaluated the expression and activity of MMPs and their inhibitors RECK and TIMP-2 in three human invasive cervical carcinoma cell lines. Two HPV-16- positive cell lines (SiHa and CaSki), HPV-negative cell line (C33A) were cultured either onto type-I collagen gel, Matrigel or plastic, in order to recreate their three-dimensional growth environment and evaluate the growth and invasion of the cells and expression of these genes using quantitative real-time PCR (Q-PCR). We also analyzed the gelatinolytic activity of MMP-2 and -9 by zymography. We found that the growth curves carcinoma cells are decreased and cells morphology are modified in ECM substrate. HPV-positive cell lines expressed higher levels of MMP-2, MT1-MMP and TIMP-2 than the HPV negative cell line. In addition, MMP-9 was expressed at very low levels in both HPV-negative and HPV-positive cell lines. We also observed that the expression of the RECK gene is higher in CaSki cells, being associated with pro-MMP-2 activity. The Matrigel substrate influence MMP-2 expression for SiHa and CaSki cells. On the other hand, we found that type-I collagen gel, but not Matrigel, can enhance pro-MMP-2 activity in all cell lines. Our results suggest that the presence of HPV is related to increased expression of MMP -2, MT1-MMP and TIMP-2 and pro-MMP-2 activity in HPV-positive cells than in HPV-negative cells.