Fe-AZT and Pd-AZT Synthesis and Effects

Fe-AZT and Pd-AZT Synthesis and Effects Synthesis and Effects of Fe-AZT and Pd-AZT on Viability of Human Hepatocytes and Hepatocellular Carcinoma Cells Submitted by: Anna Harutyunyan Introduction Cancer is one of the major causes of mortality in the world. In 2015, according to the National Cancer Institute, over 1.6 million new cases of cancer were reported in the United States. The estimated cancer deaths for the year of 2015 were over 500,000. According to the National Cancer Institute projections, in 2016 an estimated 1.7 million new cases of cancer were diagnosed in the United States and 595,690 people will die from the disease (National Cancer Institute). Although new cancer treatments and therapies are designed and implemented every year, cancer is still the number two cause of mortality in the United States, therefore developing and testing new effective anticancer agents is crucial. Hepatocellular carcinoma (HCC) is one of the deadliest forms of cancers (Venook et al., 2010). The overall 5-year survival rate of HCC is less than 17%, making HCC the fastest rising cause of cancer related death in the United States (American Cancer Society 2016; Mittal and El-Serag, 2013). The annual age-adjusted incidence rates of HCC increased from 1.4 per 100,000 individuals in 1975-77 to 4.8 per 100,000 in 2005-07. An estimated 39,230 new cases of liver cancer (including intrahepatic bile duct cancers) were expected to occur in the US during 2016, approximately three-fourths of which would be hepatocellular carcinoma. An estimated 27,170 liver cancer deaths were expected in 2016 (American Cancer Society 2016). In 80-90% cases HHC occurs with and after cirrhosis. HCCs major risk factors are Hepatitis B and C viruses, cirrhosis, non-alcoholic fatty liver disease (NAFLD), overconsumption of alcohol and exposure to other carcinogenic substances (Mittal and El-Serag, 2013). Liver ca ncer is the sixth most common neoplasm and the third leading cause of cancer-related deaths, accounting for approximately 600,000 deaths annually (Venook et al., 2010). Because of early metastasis and progression, HCCs treatment is difficult, and traditional chemotherapy has shown limited success (Sabokrough et al, 2014). A research study published in 2015 suggested that an organometallic complex of platinum (II) azidothymidine (Pt (II)-AZT) has an antitumor effect on rat hepatocellular carcinoma cells. It was shown that this complex was significantly more effective in tumor suppression than the AZT without platinum (Sabokrough et al., 2014). Several organometallic complexes of AZT (with Zinc, Cobalt, Copper and Iron) were synthesized and characterized previously. The Iron complex of AZT (Fe-AZT) was shown to be the most stable (Shirvastav et al.) and have antimicrobial activity against 4 groups of bacteria. Electron rich ligands like AZT effectively bind and interact with metal ions producing metallodrugs which offer promising therapeutic application in terms of combating the drug-resistant strains of pathogens. It is also logical that a metal ion can influence the biological activity and therapeutic efficacy of the bio-molecule they bind (Shirvastav et al.) Thus it is useful to investigate the effect of a metal ion on the efficacy and mode of action of AZT in suppressing malignant cells and inducing apoptosis. To date, no research has been published regarding synthesis of Pd-AZT the inhibitory effect of both Fe-AZT and Pd-AZT on malignant cells. The aim of this research project is to synthesize Fe-AZT and Pd-AZT, confirm their structure and molecular mass and test their effects on viability of human hepatocellular carcinoma cells and normal human hepatocytes. Background Normal cell cycle In order to understand the biology of cancer it is crucial to understand the cell cycle of normal cells and cancerous cells. All cells in a given organism are under strict control of multiple regulatory agents, such as RB or p53 that control cell growth and keep the proliferative index stable. If there is a need for the cell to divide, the proliferative genes will turn on, or the suppressor genes that keep proliferation from occurring will be shut down, which will result in cell division (Hardin et al., 2014). The normal cell cycle consists of the following phases: G1, S, G2, M. During the G1 phase duplication of organelles, membrane systems and other important components happens. There is an important checkpoint at the end of this phase referred to as the restriction checkpoint, which verifies that DNA synthesis was successful and no errors have been detected. Next is the S phase during which the cell duplicates each chromosome. During the G2 the rest of the components that were not synthesized in the G1 phase are synthesized. A checkpoint after this phase makes sure the cell is ready to divide, and no errors in chromosome duplication were made. Mitosis is the next phase, when the cell physically divides (cytokinesis) making two identical daughter cells. A checkpoint during this phase checks if both of the daughter cells received the correct number of chromosomes. After division, the cell may remain in its silent G0 phase, when it does not divide, but continues to function. If any errors are detected on any of the checkpoints, the cell cycle is put on hold and the cell will undergo apoptosis. This process is governed my many enzymes and complexes, and theoretically, malfunction of any of these enzymes may result in uncontrolled cell division/proliferation. This can be the onset of carcinogenesis. Figure 1. The cell cycle phases with their checkpoints. Cancer cell cycle It is important to note that the normal cell cycle is under control of thousands of regulatory elements that are coded for by different genes. If mutations occur in the genes that code for the key elements keeping cell proliferation at bay, one level of control over the cell cycle is lost. There are cell signaling mechanisms through which immune cells detect faulty cells, send them death signals and the faulty cell undergoes apoptosis. It takes more than one mutation for a normal cell to start behaving like a cancer cell. Usually it is either the loss of the function of suppressor genes, or overexpression of proliferative genes that result in uncontrolled cell proliferation. When the cell becomes malignant, it loses the ability to respond to death signals, thus does not undergo apoptosis. The malignant cell cycle is slower than the normal cell cycle, however, since the cancer cells keep dividing and do not die, their number grows exponentially resulting in tumors (Weinberg, 2014). There is distinct cytological difference between normal and cancer cells (Figure 2). A normal cell has a smaller, regularly shaped nucleus, a low nucleus/cytoplasm ratio, is well differentiated and has well defined borders. Cancer cells have larger, irregularly shaped nucleus, high nucleus/cy toplasm ratio, are less differentiated and have irregular borders. In contract with normal cells that adhere to each other, cancer cells are less adhesive and break away from each other (Weinberg, 2014). Figure 2. The comparison of the cytology of normal cells and cancer cells. Azidothymidine (AZT), Fe(III)AZT, Pd(II)AZT Azidothymidine (AZT, Zidovudine) is a thymidine derivative in which the 3-hydroxy group is replaced by an azido group (Figure 3). It has been shown to have antitumor effects on different animal carcinoma cells both in vitro and in vivo through inhibition of telomerase activity and by causing cell cycle arrest (Gomez et al., 2012; Hadizadeh et al., 2014, Cooper and Lovett, 2011; Matteucci et al., 2015). Figure 3. Thymidine (left), Azidothymidine (right). Since cancer cells have higher proliferation rate than normal cells, their thymidine turnover rate is higher, which could contribute to their increased sensitivity to AZT. Several studies conducted by Fang et al., have shown that a hepatocellular carcinoma cell line (HepG2) is significantly more sensitive to AZT toxicity as compared to the normal hepatocyte (THLE2) sensitivity (Fang et al., 2014; Fang and Beland, 2009). The current hypothesis on how AZT affects the cell suggests that AZT is phosphorylated intracellularly yielding AZT-triphosphate and AZT-monophosphate. The AZT-monophosphate can be incorporated into the DNA structure instead of thymidine due to its similar molecular structure and shape. However, in contrast with thymidine, AZT lacks the 3 hydroxyl group which is the group that forms phosphodiester bonds between nucleotides in the DNA backbone structure. This means, wherever the AZT phosphate is incorporated in one of the DNA strands during DNA replication, the elongat ion of that strand halts (Figure 4). This is one of the mechanisms through which AZT causes damage to DNA (Fang et al, 2014). The commercial name of AZT is Zidovudine, and it is the basis of AIDS treatments. AZT blocks the replication of HIV-1 virus by competitively inhibiting the viral reverse transcriptase (RT). In other words, HIV RT prefers AZT to normal nucleotides. As described above, AZT is phosphorylated to AZT-triphosphate and is incorporated into viral DNA, halting nucleotide chain elongation. It is also known that AZT-triphosphate can be incorporated into eukaryotic DNA, although its affinity for DNA polymerases is lower than that for RT (Gomez et al, 2012). It was shown that Pt(II)-AZT is more effective in suppressing cancer cells than AZT, thus it has been suggested that having a transition metal bound to the central nitrogen atom of the azido group increases the complexs affinity for G-C and A-T base pairs and the P-backbone of DNA (Das and Pitre, 2007; Sabokrouh et al, 2015). Materials and Methods Fe-AZT and Pd-AZT synthesis To carry out the synthesis of Pd and Fe complexes, solid AZT and a standard ion solution of each metal (iron nitrate nonahydrate (Fe(NO3)3 ·9H2O and palladium chloride (PdCl2)) will be purchased from Sigma Aldrich (St. Louis, MO). Synthesis will be performed according to the protocol by Das and Pitre (2007). A 1:1 molar ratio of aqueous solution of AZT and metal ion will be refluxed for 3 hours. The volume of the reaction mixture will be reduced by 75% in order for the complex to precipitate. The solid product will be vacuum filtered, washed twice with ice-cold water, recrystallized and air-dried overnight. Infrared absorbance spectra will recorded for the AZT, Fe-AZT and Pd-AZT complexes. In the experiment conducted by Das and Pitre (2007) the comparison of IR spectra of pure AZT and its complex with Fe, the spectrum indicated a shift in bands from 2170 to 2150 cm-1 due to complexation through N atom of azido group. A similar band shift in the IR spectrum of Fe-AZT and Pd-AZT is expected. A Matrix Assisted Laser Ioniation/Distortion Time-of-Flight (MALDI TOF) mass spectrometry analysis will be performed for the Fe-AZT and Pd-AZT to determine the molar mass and the stoichiometry of the complexes. The matrix for analysis will be composed of 1:1 saturated Anthranilic Acid and Nicotinic Acid (Sigma Aldrich, St. Louis, MO). The samples and matrix will be dissolved in 45% acetonitrile: 55% water (van Kampen et al., 2004). Cell cultures HepG2, THLE2. Liver carcinoma cell line HepG2 and normal human liver cell line THLE2 will be obtained from American Type Culture Collection (Manassas, VA). The standard protocol recommended by ATCC will be used for establishing and maintaining hepatic cell lines. The cell lines will be plated at a density of 5 x 103 cells/cm2. HepG2 cells will be cultured in DMEM with 10% fetal bovine serum and antibiotics (penn/strep). THLE2 cells will be cultured in LHC-8 medium with 70ng/ml phosphoethanolamine, 5ng/ml epidermal growth factor, 10% fetal bovine serum and antibiotics (Fang et al., 2009). After ensuring sufficient cell growth and several passages of each cell line, the two cell lines will be divided into 4 groups each: control (no treatment) and treated with each drug: AZT, Fe-AZT and Pd-AZT (Table 1). Table 1. The experimental setup of the control and experimental groups of cells. Control 1 (C1) THLE2 cell line (no treatment) Control 2 (C2) HepG2 cell line (no treatment) Experimental 1 (E1) THLE2 cell line+AZT Experimental 2 (E2) HepG2 cell line+AZT Experimental 3 (E3) THLE2 cell line+Fe-AZT Experimental 4 (E4) HepG2 cell line+Fe-AZT Experimental 5 (E5) THLE2 cell line+Pd-AZT Experimental 6 (E6) HepG2 cell line+Pd-AZT The cells in the treated groups will be further categorized by the concentrations of the drug. HepG2 cells will be incubated with 2, 20 or 100  µM aqueous solution of AZT, Fe-AZT or Pd-AZT for 14 days. THLE2 cells will be incubated with 50, 500 or 2500  µM aqueous solution of AZT, Fe-AZT or Pd-AZT for 14 days. Each group of cells will be seeded at 5 x 103 cells/cm2 density in 6-well plates. The cells will be passaged weekly during the two-week treatment period. The dosage and incubation time were chosen based on a similar study (Fang et al., 2013; Matteucci et al., 2015; Sabokrouh et al., 2015). Cell viability assay After 14-day treatment period, 103 106 cells from each group will be seeded in a 96-well plate, incubated for 4 hours, and treated with MTT reagent, followed by a 8-12 hours incubation at 37 °C. After the incubation with MTT, when the cells have metabolized the yellow tetrazolium dye (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to purple formazan, the cells will be treated by a detergent to release the formazan into the solution, and the absorbance will be measured at 570nm using an ELIZA reader. The number of metabolically active cells will be determined using a previously made calibration curve. The statistical differences will be calculated using a Two-Way ANOVA and Tukeys Post-Hoc Test. Anticipated outcomes. The expected mass-to-charge ratio from mass spectrometry analysis of Fe-AZT complex is 323.1, that of Pd-AZT is 373.6. Based on the published literature and the adopted hypothesis, all three drugs are expected to decrease cell viability. The concentration of either drug should have positive correlation with decrease in cell viability. Based on previous observations, it is expected that the HepG2 cells will be significantly more sensitive to both complexes, thus will be significantly less viable after the treatment compared to the THLE-2 cells. Study Participants and Timeline Anna Harutyunyan is the primary author of this study. She is currently a senior biology and chemistry major at Wilson College. This project will serve as Annas senior research project. She will conduct the research and present the results with the supervision of her advisors Dr. Deborah S. Austin (Sponsoring PAS member) and Dr. M. Dana Harriger (PAS Member). The research study began in September 2016 and will be completed by April 2017. Annas research findings will be analyzed and written as her senior thesis. Anna will also present her findings at the 2017 Pennsylvania Academy of Science meeting and at the Wilson College research colloquium. Budget Item Price MTT Assay Kit (ThermoFisher) $235.00 Zidovudine (Sigma) $124.00 LHC-8 medium (Gibco) $140.00 Total $499.00 Facilities and Equipment The MALDI TOF mass spectrophotometer will be provided by Pennsylvania State College of Medicine Mass Spectrometry Facility under supervision of Bruce Stanley, PhD. All materials, other instruments, and equipment not listed, including cell culture media and supplies, will be provided by the Department of Physical and Life Science of Wilson College. References       American Cancer Society. Cancer Facts Figures 2016. Atlanta: American Cancer Society; 2016. Bilsland AE, Stevenson K, Liu Y, Hoare S, Cairney CJ, Roffey J, et al. 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