An Evaluation of Novel Anti-Cancer Agents:
Platinum and Palladium (IV) Complexes

by Evangeline Easterly

Figure 1[Pt(phen₃)]⁺⁴

ABSTRACT

Platinum compounds have demonstrated cytotoxicity against a variety of cancer cell lines. Cisplatin, in particular, has been used effectively as a treatment for human cancers and is used alone or in conjunction with other cancer therapy agents. In spite of its efficacy, there are many problems with using cisplatin, including side effects, development of resistance, and the need for intravenous administration. A new metal compound, a diimene complex of platinum [Pt(phen₃)]⁺⁴ , has been tested for effects against cancer cells and found to be cytotoxic. After this initial compound was found to be promising, analogus compounds with Pt and Pd metal centers and additional diimene ligands were synthesized for cytotoxicity testing. In order to determine differences in the effects of the type of metal ligand and the number of ligands on Pt and Pd, novel complex ions [Pd(dione)]⁺⁴, [Pt(dione)]⁺⁴, and [Pt(dione)₂]⁺⁴ were tested for cytotoxicity against a variety of normal and transformed cell lines to study the in vitro effects of the metals and their ligands. Photographs were also taken so the mechanism of how these compounds treat cells could be better understood. Results indicated that the [Pd(dione)]⁺⁴ is an overall effective anticancer agent, and cells appear to undergo cytotoxicity, cytostasis, and necrosis when exposed to [Pd(dione)]⁺⁴, [Pt(dione)]⁺⁴, and [Pt(dione)₂]⁺⁴.

I. INTRODUCTION

Metal compounds were first demonstrated as having antitumor effects when studies by Dr. Barnett Rosenberg confirmed that cisplatin was effective against a variety of tumors. Over the past 25 years, pre-clinical screening of several thousand new molecules based on platinum complexes have resulted in the identification of 28 compounds that have entered clinical development. Since the original testing of cisplatin, the compound has been used either alone or in combination with other cancer therapy agents. In spite of its success, there are also side effects from using cisplatin as well as from other chemotherapeutic agents. Thus, there is continual research to develop more platinum (Pt)-based compounds based upon these cisplatin studies. The first homoleptic diimene complex of Pt (IV) has been synthesized and it, along with a variety of similar Pt and palladium (Pd) compounds, have been shown to be cytotoxic to cells. Based on previous research, these novel Pt and Pd compounds need further investigation.

A. CANCER

Cell communication is important in the body so that cells may respond to stimuli and changes. The cells’ response to extracellular stimuli is the result of a complex network of signal transduction pathways that coordinates cell division and differentiation. When normal cells lose the ability to respond to stimuli in signaling pathways, they give rise to tumors. That is, the failure to regulate proliferation and differentiation results in uncontrolled growth of cells. Tumors can either be benign, remaining localized and not spreading to other tissues, or malignant, a mass of rapidly dividing cells which can spread to adjacent tissues. Malignancy is commonly termed cancer. The lack of regulated cell growth is due to the expression of oncogenes and the inhibition of tumor suppressor genes, which produce specific regulatory proteins for the cell. Scientists have studied anticancer agents and their mechanisms in the cell in order to find the cure for cancer.

B. OXIDATION STATES OF ANTITUMOR AGENTS

The oxidation states of metal compounds pose an interesting question because they vary, depending on the structure of the compound and the compounds’ reaction during antitumor activity. Platinum has two dominant valences or oxidation states, +2 which forms a square planar complex, and +4 which forms an octahedral complex. The anticancer activity of Pt(IV) compounds has proven to be successful, but there is no confirmation as to whether or not these compounds are active in the +4 or +2 oxidation state. Blatter et al. suggested that some Pt(IV) compounds may act as prodrugs, which are inactive forms of a drug that can be converted into the active form by a metabolic process. The Pt(IV) compounds may need to undergo metabolism or reduction to Pt(II) in order to activate antitumor properties. Some of the prodrugs are reduced to trans-agents and not cis-agents, so they are not able to act like cisplatin. However, there are reports of Pt (IV) complexes not undergoing reduction and entering the cell as Pt(IV) and damaging DNA.^, The fact that there are many different responses of Pt(IV) means that Pt (IV) complexes are an excellent avenue for research.

C. NEW SYNTHESIS

Cisplatin is the best model we have for anticancer therapy; we know what it attacks, the tumors it is most effective against, and the adverse side effects. By studying cisplatin and other metal compounds, researchers are trying to develop more antitumor drugs. Dr. Robert M. Granger in1996 synthesized the first homoleptic diimene complex of Platinum (IV), [Pt(phen₃)]⁺⁴.³ [Figure 1] Dr. Granger got the idea to make this compound when previous studies had indicated that some Ru(II) and Ru(III) complexes bound to DNA. Since Ru interacted with DNA, he thought other metal compounds, such as Pt and Pd, would interact with DNA, as well. His early grant proposals are targeted to site-specific DNA binding.

The synthetic scheme that he developed allows a variety of ligands to be added to both Pt(IV) and Pd(IV). Some of the ligands that can be added include 5,6-dione-1,10-phenanthroline ("dione"), dipyridylphenazine ("dppz"), 2,2’-bipyridine ("bipy"), and phenanthroline ("phen"). [Figure 2*]

These ligands are multi-ring, planar structures with nitrogen bases.

Dr. Robin Davies and Margaret Dally ’99 of Sweet Briar College, completed studies with Pt(IV) and Pd(IV), in comparison with the Ru compounds, and showed that Ru was less toxic to the cells. These newly synthesized complexes were very interesting due to their octahedral structure and Ru compounds of similar structure have been shown to interact with DNA. ^,

In order to investigate the mechanism of action of Pt(IV) complex ions, their DNA binding characteristics were studied. In a DNA viscosity assay, the [Pt(phen₃)]⁺⁴ interacted with DNA and caused viscosity greater than the normal unbound DNA. This showed that Pt(IV) was intercalating in the DNA by making the DNA coil. The Pt(IV) compound attaches between base pairs, extends the DNA ladder, and probably would disturb the structure of the DNA so it could not be replicated properly. An agarose gel electrophoresis showed retarded motion of pBR322 DNA in the gel, indicating that Pt(IV) was bound to the supercoiled pBR322 DNA. This indicated that the Pt(IV) was groove binding, or binding to one of the major or minor grooves within the DNA, increasing bending of the DNA. This contrasted the results of the viscosity assay. These tests indicated that the [Pt(phen₃)]⁺⁴ bound to DNA, however, both suggested two contrasting pieces of data, either intercalation or groove binding, on the DNA interaction with [Pt(phen₃)]⁺⁴ . The current model suggests that Pt(IV) is ‘intercal-kinking’ or undergoing partial edge-intercalation. The Pt(IV) is neither groove binding nor intercalating, but binding to the DNA structure causing a bend or kink in the DNA. More studies need to be done to determine how the Pt/Pd functions in the cancer cells as well as how it interacts with DNA.

D. PAST RESEARCH

The original compound, [Pt(phen₃)]⁺⁴ , was tested in cell cultures because of the DNA binding activity and the fact that other Pt(IV) compound were known to have anticancer activity. Both normal and transformed (infected with the Rous sarcoma virus) chicken embryo fibroblasts (SL-29, from the American Type Culture Collection, ATCC) were exposed to [Pt(phen₃)]⁺⁴. The cells were exposed to a variety of concentrations and preliminary results suggested that at concentrations of 1 x 10^–5 M [Pt(phen₃)]⁺⁴ was toxic to transformed chicken embryo fibroblasts. There was at least 70% growth suppression of transformed cells as compared to normal cells, which meant there might be selective cytotoxicity towards the transformed cells.

These results led to more tests using the murine lymphocytic leukemia cell line L1210 (ATCC) which has been used in a variety of tests with Pt(II) and Pt(IV) compounds. ^, Cells were exposed to 1 x 10^-5 M [Pt(phen)₃]⁺⁴ , [Ru(phen)₃]⁺², and [Ru(dipy)₃]⁺² for 68 hours. Only [Pt(phen)₃]⁺⁴ showed appreciable toxicity. Additional testing of HL-60 (ATCC) human leukemia cell line was conducted in which cells were exposed to 1 x 10^-5 M Pt(dipy)₃]⁺⁴, [Pt(dione)]⁺⁴, Pt(phen)₃]⁺⁴, [Ru(phen)₃]⁺², [Pd(dione)]⁺⁴, [Pt(dione)(phen)₂]⁺⁴ and [Pd(dione)(phen)Cl₂]⁺² for 68 hours. The cytotoxicity of these compounds was determined by Margaret Dally ’99, a summer research student at Sweet Briar College. Results determined that the Pd(IV) compounds were the most toxic and that both Pt(IV) and Pd(IV) diimene complexes should be further tested.

Continuing research has worked with additional compounds and a variety of cells lines. Marisol Laserina ’99, a Senior Biology Major, tested [Pt(dione)₃] against CCD-27sk, a normal human skin fibroblast, and HT-1080, a human fibrosarcoma line. Her results indicated that there was significant cell death, with an IC₅₀ (inhibitory concentration of 50% of the cells) for both cell lines at concentrations between 10 - 100m M.

For my Junior Honors Thesis I examined the effects of [Pd(dione)Cl₄]⁺⁴ against a matched pair of normal and transformed breast cell lines, HTB-125 and HTB-126, derived from the same cancer patient. Trials demonstrated that there was selective toxicity for breast cancer cells with an IC₅₀ of 1x10^-4 M to 1x10^-5 M. The range of cytotoxicity is between these two concentrations. This compound needed to be tested against a variety of cells lines to look at the effects.

Additional tests were performed by Vanessa Corry ’01 and Agnes Saba t’01, both summer research participants at Sweet Briar College. They looked at three cell lines, WI-38, a normal lung cell line, A-427, a transformed cell line derived from a carcinoma of the lung, and SW-480, a transformed cell line derived from an adenocarcinoma of the colon. These cell lines were tested against [Pd(dione) Cl₄]⁺⁴ as well as [Pt(dione) Cl₄]⁺⁴ , [Pd(dione)₃]⁺⁴ , and [Pd(dppz) ₃]⁺⁴. Results indicated there was selective toxicity of Pd mono-dione towards A-427 lung cancer, with an IC₅₀ of 1m m, but only 50m m towards SW-480. The Pt mono-dione was cytotoxic from 100 to 10m M, but not selectively toxic. At 100m M the Pd tris-dione was selective against the A-427, but was not as toxic towards the SW-480 colon cancer line. Pd tris-dppz appeared to be selectively toxic at 10uM towards the SW-480.

Overall, the Pd mono-dione appears to be the most promising compound, showing selective activity against breast and lung cell lines with at IC₅₀ of 5 m M and 1m M, respectively. The Pd tris-dione and Pd tris-dppz, were respectively active against A-427 at 100m M and against SW-480 at 10m M. The most successful compounds to date seem to be the Pd(IV) complexes.

E. Pt/Pd CELL TOXICITY STUDIES

1.Ligands

The purpose of comparing a variety of Pt and Pd compounds is to study the potential differences between the metals centers of the compounds, the structure of each ligand, and the level of substitutions. The compounds may interact in a variety of ways towards cells. This information will help determine the cytotoxic results of a variety of ligands and metals on the same cell lines as well as toward a variety of cell lines. The metal centers differ in molecular weight, Pt 195.08 kD and Pd 106.5 kD. The structures of the ligands and number of ligand substitutions should determine the binding affinity of the molecule towards its target, which is still unknown. Preliminary studies have recognized the capabilities of newly synthesized Pt(IV) and Pd(IV) diimenes for cytotoxic activity.

The purpose of the study was to evaluate the potential anticancer effects of additional compounds, Pd(dione)]⁺⁴ Pt(dione)]⁺⁴ , and[Pt(dione)₂]⁺⁴, by cell-culture-based testing. I also compared the difference in effect of compounds with the same metal center, Pt, with one and two ligand substitutions. These will be discussed below.

2. Cell morphology

Normal cell lines have a finite life span so that they can only undergo a certain number of cell divisions before genetic cell death occurs. They also grow in monolayers by exhibiting contact inhibition, so that if cells touch one another, signals are sent out so that neighboring cells will stop dividing in G₀ phase. Transformed, or tumor cell lines, occur from genetic mutations and result in irregular growth. Tumorgenic cells lack contact inhibition so they form layers of cells and reach a higher density than normal cells.

After cancer cells are exposed to antitumor agents, the cells can resist the compound, stop proliferation (cytostatic response), or die (cytotoxic response). Cell death can occur by one of two ways, apoptosis or necrosis. The appearance of the cell can tell us how the cell is dying. Apoptosis is programmed cell death in which nuclear chromatin condense and cell size is greatly reduced. Necrosis results from any trauma towards the cell. The cells increase in size, there is formation of lysosomes and vacuoles, and the membranes undergo a massive disruption. It is important to compare the appearance of the cells and the cell viability after exposure to the compounds because this should determine the effect of the compound. Photographs of the cells after exposure to the compounds and the cell viability graphs will help to determine the how these compounds are killing the cells.

II. RESULTS

A. TOXICITY STUDIES

Three compounds, [Pd(dione)]⁺⁴, [Pt(dione)]⁺⁴, and [Pt(dione)₂]⁺⁴, were tested against a range of cell lines: 1) normal mammary gland fibroblasts (HTB-125) and cancerous mammary gland epithelial cells (HTB-126) from the same individual, 2) A-427, a lung carcinoma cell line 3) WI-38, a lung fibroblast cell line and 4) HT-1080, an epithelial cell line from a fibrosarcoma. At least two trials were done for each cell line and compound, all of which were run with a concentration of 1 x 10^-5 M of the compounds. The normal cell lines are indicated by graphs with diagonal lines whereas the cancer cell lines are indicated by black graphs.

Exposure to [Pd(dione)]⁺⁴ (Pd mono-dione) in figure 3 shows tremendous cell death, 87%, for HT-1080. The A-427 and WI-38 showed similar toxicities from 43-46%. There is a large difference between the matched cell lines of HTB-126 and HTB-125, so » 39% more of the cancer cells are dying when exposed to Pd mono-dione than the normal cells.

Observing the percent viability after exposure to [Pt(dione)]⁺⁴(Pt mono-dione) in figure 4, three transformed cell lines were compared against one normal cell line. In the matched set of cell lines, A-427 and WI-38, there is 15% difference between viability rates, and HT-1080 is 20% less viable than the WI-38. HTB-126 had a 15% higher survival rate than the WI-38, and 36% more HTB-126 survived than HT-1080. The HT-1080 had the lowest viability rate when exposed to this compound. In figure 5, the HT-1080 show 80% cell death in [Pt(dione)₂]⁺⁴ and these were the most affected cell lines exposed to this compound There was 45-50% cell death for the A-427 and WI-38.

Overall, the response of each cell line to all the compounds is important. For example, the HT-1080 is very responsive to all the compounds, showing from 12-29% survival. The Pd mono-dione is the most effective with a survival rate of 12%, and the Pt di-dione, 20% survival, is more effective than the 29% survival of cells exposed to Pt mono-dione.

The A-427 were consistent in the range of cell survival, 44 -59% against all the compounds. Mono-dione survial differed less than 1% between the metal centers, they both had » 45% cell survival. The effects of the numbers of ligands for the Pt compounds varied only 5 % from the Pt mono- to the di-dione, 45-50% respectively. Therefore, the mono-diones resulted in slightly more cell death than the di-diones.

After exposure to the three compounds, the WI-38 normal cell line shows little variation in cell survival rates, 48- 55%. Cell survival results indicate that the mono-diones were at least 4% more successful in cell death than the Pt di-dione. Pd mono-dione had 47% cell survival, which was close to the 49% cell survival after exposure to Pt mono-dione. The HTB-126 survival rate was 50% for the Pd mono-dione and 65% Pt mono-dione. The Pd produced 15% more cell death than the Pt.

After testing three different compounds, Pd mono-dione, Pt mono-dione, and Pt di-dione, the Pd mono-dione was the most effective in cell death for all of the cell lines. For cell lines A-427, WI-38 and HTB-126, the Pt mono-dione was then found to be more effective at cell death than the Pt di-dione. The results for the HT-1080 suggested that the Pt di-dione was more effective at cell death than the Pt mono-dione.

      

III. DISCUSSION

A new innovation in antitumor drug screening is to show the difference in cell survival rates between a matched set of cell lines. As proven from many previous studies in the literature, most anticancer agents bind to DNA in massively proliferating cells, inhibiting replication of cells. However, those studies do not compare the effects of anticancer agents on normal cell lines. Research on anticancer agents should be done on both transformed cells and their normal equivalent because the normal cells of the body as well as the cancer cells will be exposed to the anticancer agent upon administeration. There are normal cells in the body that have a high proliferation rate, and the anticancer compounds usually affect these highly proliferating normal cells. The binding of anticancer agents to normal cells causes side effects such as nausea and nephrotoxicity. Due to the side effects of metal anticancer agents on normal cells, it is important to study the compound's effects on normal and cancerous cells.

Within this study, comparisons were made between newly synthesized Pt (IV) and Pd (IV) compounds. Comparisons were made between the effects of the metal centers with the same ligand, and the effects of a single metal with varying numbers of ligands. Overall, the results from the three compounds, Pd(dione)]⁺⁴ Pt(dione)]⁺⁴ , and[Pt(dione)₂]⁺⁴, show that Pd mono-dione is more cytotoxic than the Pt mono-dione and Pt di-dione. None of the compounds produced a large difference between the matched cell lines of WI-38 and A-427, so perhaps more studies should be done with compounds on this cell pair. The Pt mono-dione produced varying results, ranging from 29-65% cell survival in four cell types. The Pt di-dione had different effects upon the two transformed cells and had survival rates ranging from 20% to 50%.

Microscopic observations of the cells can indicate the effect of the compounds on the cells (cytostatic or cytotoxic), and the cell death response of the cells to varying compounds. However, it is important to study the relationship between the viability assays and the cell appearance to determine the effects of compounds and cellular responses.

The HT-1080 cell line shows a perfect example of anticancer compounds producing two contrasting effects, cytostasis and cytotoxicity. Cytostasis results when compounds do not allow cells to replicate but remain at the same cell number until the cells die. Cytotoxicity is when compounds cause an increased rate of cell death, usually caused by damaging the DNA. When comparing the appearance of HT-1080 exposed to both Pt mono-dione and Pt di-dione, the percent viable cells does not necessarily correlate to the appearance of the cell lines. The HT-1080 in exposed to Pt mono-dione are extremely round and detached from the flask, indicating that they will undergo necrosis. These cells appear less viable and closer to cell death than the HT-1080 exposed to Pt di-dione.

Exposure to Pt di-dione causes the cells to partially round, looking almost normal. By studying the shape and amount of detachment for the cells, it seems that the cells exposed to the Pt mono-dione would be less viable than cells exposed to Pt di-dione. However, this is not the case. Exposing HT-1080 cultures to Pt mono-dione shows fewer viable cells than those exposed to Pt di-dione. The mechanism for the correlation may be that Pt di-dione is a cytostatic compound which causes cells to partially round up and does not attack the DNA. Cytostasis, the inhibition of proliferation, would account for the decreased percentage of viable cells as compared to exposure to the mono-dione. The Pt mono-dione is most likely cytotoxic to the cells and causes them to undergo necrosis. Another factor influencign these results could be the length of time the cells were exposed to the compounds. The viability assays were exposed only 2 days whereas the photographs depict the cells after 3 days of exposure.

The A-427 exposed to the Pd and Pt compounds produce cells with increased vacuole formation and fragmentation, characteristics of necrosis. Cells exposed to Pt di-dione seemed to be sparser with fewer cells attached to the flasks than the cells exposed to Pt mono-dione. The Pt di-dione exposed cells were stained two different ways, by using methylene blue, which binds to nuclei, and Giemsa, which binds to proteins. From the two stains, we can clearly see the vacuole formation and fragmentation from the methylene blue and the shape of the cytoskeleton by the Giemsa. The fragmentation is a result of necrosis. Visual images using different staining techniques may provide evidence to cell shape, as seen by the methylene blue stain, and physical characteristics of the cell, as shown by the Giemsa stain.

The pictures of WI-38 seem to show that more cells die when exposed to the mono-diones as opposed to the di-dione. Cells may have a 5% survival rate upon exposure to the di-dione. The appearance of the poorly intact WI-38 cells in the pictures somewhat correlates with the low cell viability rate. The cells exposed to the mono-diones appear rounded, have no apparent nuclei, show increased vacuole formation, and have breached membranes, all qualities of necrosis. Breached cell membranes, and fixture of the cytoskeleton to the flask is enough evidence to say that the mono-diones kill these cells by necrosis.

Comparing the picture of cells exposed to Pt di-dione and the percent viability graphs, the cells seem to undergo cytostasis. Only a slight amount of cells detached during the time of exposure to Pt di-dione, and the percent viable cells compared to unexposed controls showed 55% cell survival. The cells exposed to Pt di-dione do not look like there was a 45% decrease in viability because many of the cells were still attached to the flask. There appears to be a ‘visually’ higher amount of living cells than is reflected in the viability graph. The low percent viability rate is due to the cytostatic compound, which causes the cells to cease replication upon exposure to Pt di-dione.

The HTB-126 breast cancer cells sdeem to undergo apoptosis in response to the Pt di-dione. This indicates that the structure of the Pt compounds, either with one or two ligands, can have varying responses when exposed to different cell types.

IV. CONCLUSION

A. OVERALL RESULTS

The success of cisplatin led to the discovery of a new class of anticancer agents: metal coordination complexes. This discovery initiated the hunt and race to find more metal anticancer complexes. Research information about DNA damage and resistance may help us understand the mechanisms by which the compounds act and lead to developing better metal anticancer agents. Despite trials for numerous metal compounds, platinum-containing compounds continue to be the most widely used and explored as potential chemotherapeutic agents.

When comparing the results from this experiment, we can see a variety of cell lines that have been exposed to a wide range of compounds. This preliminary level of research has increased the amount of knowledge we know about this new class of anticancer agents. [Figure 33] In addition, I have begun an investigation into the mechanisms of actions of the Pt(IV) and Pd(IV) compounds.

Figure 6 is a table of cell lines tested against compounds made by Dr. Granger to date. The table does not include the preliminary studies made by the Sweet Briar College Research Team. The cell lines and compounds used in this study’s experiments are highlighted in gray. There is still a large portion of the table to be explored in addition to synthesizing more compounds and testing them against more cell lines.

The effects of the compounds on the different cell lines suggests that each compound behaves differently towards different cells. Each cell line reacts differently to antitumor agents because they all have varying mechanisms of action to repair them once exposed to heavy metal compounds. Varying mechanisms may be a result of the involvement of proteins within cells, such as HMG or repair proteins as mentioned earlier. Likewise, as shown by these results, the metals and their varying ligands behave differently once they enter into cells; however, the mono-diones seem to be the most successful. This means that each compound should be tested against a range of matched cells (normal and tumorgenic) in order to find the best antitumor compound for that cell type.

B. FUTURE RESEARCH

Due to the varying effects of the compounds, they should be tested on a variety of cell lines, and determine the most effective concentration of the compounds. In addition, the counter ions of the complex ions should be assessed for cell death. It is also important that new compounds, such as Pd[dione₂]⁺⁴ and Pd[dppz₂]⁺² , be tested for cell death rate against HL-60 because preliminary cell death rates for various compounds have been completed with this cell line. A measurement of the time course of cell death after exposure to these compounds would be important to see if the Pt/Pd might be incorporating or binding to the DNA like most anticancer agents. This could be done by counting the cells at hourly intervals after exposure. If many cells die within three hours of administering the compound, then we can tell the phase of the cell cycle in which the compounds are killing the cells. It would also be interesting to determine the effects of Pd di-dione and other Pd compounds with single substrates.

Looking at the mechanisms of action for these compounds is important because as described earlier, the compounds have been shown to have varying effects on the cells. By studying the mechanisms of action we can determine the oxidation state, target structures for these molecules, and their potential binding effects on matched pairs of cells or DNA. This may be done by means of cell fractionation techniques and NMR or AA studies. A gel electrophoresis and viscosity assay, similar to what was described previously, would give us indications as to how these various compounds are binding or attaching to DNA.

Aside from the mechanism of action of anticancer agents, it would be very interesting to study tumor resistance. This could be done by letting cell lines that have already been exposed to anticancer agents grow to confluency. These secondary cells are resistant to the mechanism that killed the previous cells. So, another compound, which may have a different mechanism of action towards that cell line, could be administered. Using a different compound, one that the cell is not resistant to, may provide a way to treat the resistant cells. By using a series of different anticancer agents with different mechanisms of action, a combination of compounds might be able to inhibit growth and resistance of the tumor.

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*Figure 2 not available


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