Synergistic Enhancement of Anti-Cancer Activity of Cisplatin by Noscapine, an Opioid Alkaloid, in Human NonSmall Cell Lung Cancer

Pratik Sachdeva and Bishoy Ameen

CONTENTS

CONTENTS

Contents
1 Abstract 2 Introduction 3 Materials and Methods 4 Results 5 Discussion and Conclusion 6 Bibliography 3 4 6 10 13 15

ABSTRACT

Abstract
Human Non-Small Cell Lung Cancer (NSCLC) is often treated with Cisplatin, a platinum chemotherapeutic

agent. Despite its moderately high ecacy, Cisplatin has rather dangerous side eects including nephrotoxicity and neurotoxicity due to the nature of its mechanism. This study examines a possible synergism between Cisplatin and an opioid alkaloid, Noscpaine, in hopes of improving the ecacy of Cisplatin and restraining its cytotoxicity to normal cells. Chemotherapeutic eectiveness was measured in H460 and A549 NSCLC cell lines by means of an isobolographic method with a standard cell plate study. The cell plate study treated the cells with various combinations of Noscapine and Cisplatin in 200L wells of 96well plates. The cogency of the drugs in terms of reduction of tumor volume through the cell plate study was determined with an absorption spectrophotometer. With the cell viability data, we determined the combination index values of Noscapine and Cisplatin which numerically describe their chemical relationship. Combination indices of .31 .05 to .57 .04 were obtained which indicate synergistic to strong synergistic eects of the Noscapine and Cisplatin combination treatment. Furthermore, it was found that this treatment reduced tumor volume by 78.1 7.5% with respect to the control compared with 38.2 6.8% reduction by Cisplatinum and 35.4 6.9% reduction by Noscapine alone. The study shows a clear synergism between Noscapine and Cisplatin. After further analysis of the individual mechanisms of the drugs and their eect on cell apoptotic pathways, it may be possible to use Noscapine in addition to Cisplatin for a highly ecacious and riskless treatment of cancer.

INTRODUCTION

Introduction
Lung cancer is one of the most dangerous types of cancer, killing more than 160,000 people annually

more than breast, colon, and prostate cancers combined. With lung cancer having a 5 year survival rate of roughly 15%, the need for an eective and secure treatment of this ailment is highly necessary. There are two main types of lung cancer small cell lung cancer and nonsmall cell lung cancer (NSCLC). NSCLC accounts for more than 85% of lung cancers in patients; due to this lopsided distribution and dierences between the mechanisms of the two lung cancers, this study focuses strictly on NSCLC cell lines. Generally, chemotherapy with some combination of several types of drugs gemcitabine, taxanes or vinorelbine, and a platinum drug is the rst choice treatment in NSCLC. Cisplatin (Cis H6 Cl2 N2 P t), a platinum drug known as the penicillin of cancer, is one of the more commonly used platinum drugs in chemotherapy for NSCLC. The mechanism of Cis involves induction of apoptosis by altering the DNA of the cell with the aid of an HMG protein. Cis generally enters the cell by means of active transport, but often the compound passes through the plasma membrane by passive diusion. Once in the nucleus, Cis undergoes hydrolysis due to a very low concentration of chloride ions, and Cis loses a chlorine ligand in exchange for a water molecule. At this point, Cis targets two consecutive guanines in the DNA and binds to the nitrogen atoms, because nitrogen balances the platinum charge more eectively. The Cis-guanine complex, referred to as an adduct, produces a bend in the DNA which allows the binding of proteins which contain HMG. While the tightly bound HMG along with a phenyl group of phenylalanine 37 initiates the unstacking of nucleotide bases, the DNA helix becomes kinked. Hence, the DNA is not repaired properly, and the cell dies. While Cis has been shown to successfully kill various lung cancer cell lines, its ecacy in terms of prevention of cytotoxicity has been questionable if not worrisome. Cis has a multitude of secondary eects, ranging from anemia, nausea, vomiting, neurotoxicity, and nephrotoxicity. Such side eects limit the patients quality of life and can even be life threatening in extreme cases. Furthermore, despite recent advances in chemotherapy, response rates in NSCLC are less than 50%; for those with stage IV disease, the 2-year survival rates is less than 20%. Hence, in order to optimize the chemotherapy treatment of NSCLC, a search has ensued for novel anticancer agents with nonoverlapping mechanisms with Cis that have enhanced ecacy and constrained side eects. One such compound of interest is Noscapine, an opiod alkaloid. Microtubuleinterfering agents have long been useful in cancer therapy; such examples are the taxanes and vinca alkaloids. Due to the ever evolving nature of cancer, the eectiveness and use of these agents have been restricted by drug resistant cells. Noscapine (Nos C22 H23 N O7 ), a nonaddictive derivative of opium that has long been used as an antitussive (cough suppressant), has been shown to possess similar microtubuleinterfering properties. Nos was found to inhibit cell proliferation in a wide variety of cancer cells including many drug resistant variants, while evading non-aicted cells. This quality of tremendously accurate precision has placed

INTRODUCTION

much attention on the potential of Nos; it has been found to be eective against melanoma, ovarian, lymphoma, human myelogenous leukemic, brain glioblastoma, and breast cancers. Furthermore, Nos displayed little or no toxicity to the kidney, heart, liver, bone marrow, spleen, and small intestine in previous in vivo experiments performed on mice. The success of Nos in reduction of tumor size and its paucity of cytotoxicity to healthy cells gives much hope to the improvement of cancer therapy. However, a possible synergistic relationship between Nos and another anticancer agent has not yet been explored and may oer the possibility of eective management of cancer and thereby reduce dose and associated side eects. The need to test Nos in combination with other anticancer agents is not only reasonable but also necessary; such studies could unlock to doors to advanced and highly chemotherapeutic treatments of cancer. The typical rationale would be to test Nos along with a generally accepted chemotherapeutic agent, as it clearly becomes evident after experimentation if any synergistic qualities ensue through a combination study. Consider Cis discussed above; Cis, although demonstrating ecacy in cancer treatment, has clear limitations in its mechanism and could use a great deal of improvement. While Cis is the generally accepted treatment for NSCLC, it has been shown to perform synergistically with other various anticancer agents. Hence, it would seem logical to test Cis along with Nos in order to further examine the anticancer ability of Nos and observe any possible synergisms that may arise. Both Nos and Cis have dierent mechanisms of action, hence, an isobolographic study may lead to a possible additive anticancer eect. Thus, the objectives of this study are to observe and analyze the eects and of Cis and Nos on NSCLC H460 and A549 cell lines in vitro and critically assess their cytotoxic ability with an isobolographic method, that is, testing various concentrations of Cis and Nos in conjunction and determining the combination index of the drugs. The in vitro method will be a standard cell culturing experiment with cell plates. In consideration of the above research, we hypothesize that a combination treatment of Nos and Cis will produce at least additive antitumor activity through independent mechanisms, if not synergistic anticancer capabilities. This would mean having a combination index of less than 0.6 which will be determined with the IC50 values of Cis, Nos, and the Cis/Nos combination. Hence, Cis and Nos were tested at various dilutions in 96 well plates with H460 and A549 cell lines and the percent of cancer cells killed were found. Hence, using a graph of percent cell killed vs. concentration, we were able to determine the IC50 value through interpolation. We hope to nd combination indices of a Cis/Nos study at Nos = 10, 15, 20, 30, 40 and 50 L. It is through this experiment we hope to discover a synergistic relationship between the drugs Noscapine and Cisplatin in terms of anticancer activity.

MATERIALS AND METHODS

Materials and Methods

Materials. As outlined above, the drugs of interest are Noscapine and Cisplatin, both purchased from Sigma Chemicals, St. Louis, MO. For this experiment, the NSCLC cell lines H460 and A549 were tested with the aforementioned drugs. H460 and A549 cell lines were obtained from the American Type Culture Collection. The H460 cells were grown in RPMI 1640 medium and were supplemented with 10% fetal bovine serum (FBS) in order to aid with the experimental process and provide nutrients to cells. A549 cells were grown in F12K medium and were also supplemented with 10% FBS. In order to prevent fungal growth, an antibiotic antimycotic solution of PNS (penicillin 5000 g/mL, neomycin 0.2 mg/mL, and streptomycin 0.1 mg/mL). The cells were incubated at 37 C in the presence of 5% CO2 air. A complete list of materials is indicated below. H460/A549 Cell Lines Respective Medium for the Above Cell Lines Chemical Hood Cell Incubator Cell Flasks 25mL, 10mL, and 5mL Pipettes Pipette Gun Microscope Cell Counting Apparatus PBS (Phosphate Buer Serum) Trypsin Trypan Blue Centrifuge Tube Centrifuge 96well Plates .25% Glutaraldehyde .1% Crystal Violet Sodium Dihydrogen Phosphate Quant Absorption Spectrophotometer Electronic Scale 6

3 Kimwipes Gloves Lab Coat Methods. Phase 1. Subculturing.

MATERIALS AND METHODS

The number cells obtained through purchasing is quite a miniscule amount; before the experimental process can begin, the cells must be allowed to multiply and grow in a ask with medium to have a sucient amount of cells to test. Initially, the cells were placed in a 25 mL ask in their respective medium (either H460 or A549 cell lines). The cells were kept in incubation for several days in order to allow growth and ultimately conuency throughout the ask base. When the cells grew to full conuency in the ask, subculturing was executed in order to allocate the cells throughout several asks. Subculturing is a process which utilizes a chemical called trypsin that relinquishes the cells form the ask and allows us to distribute the cells as needed. First, the asks were emptied of their medium. Then, in order to displace any extra residue that may coat the cells, the ask was washed twice with 10% Phosphate Buer Serum (PBS). Afterwards, about 5mL of Trypsin was alloted to each ask, and the asks were incubated for about 5 minutes. After incubation, the cells were checked under a micrscope to see if detachment took place. If needed, mechanical forces (comprised of tapping, at most) were used to detach any cells. Then, fresh medium was added to the cells and the newly formed mixture of trypsin and medium was poured into a centrifuge tube. In order to separate the cells from the undesired mixture, the tube was then placed in a centrifuge at 3000 RPM for about 7 minutes. Afterwards, a pellet had formed along with its supernatant. Once the supernatant was supplanted, it was replaced with fresh medium and a cell suspension formed, which could be distributed among new asks. Subculturing is essentially a prologue to the actual experiment; a direct result of subculturing is the creation of a cell suspension. The benet of this is that cell suspensions are much easier to work with the cells are more exible in that they can be transferred to a variety of placeholders, whether it be in a ask or in a 96well plate for experimentation. Phase 2. Counting The Cells. In order to compare the eects of the drug on the cell in the 96 well plates, we must be reasonably sure as to the constancy in the number of cells in each well. Hence, we must determine the number of cells in the solution and perform a dilution to obtain a desired amount of cells. To do this, an apparatus specically designed for counting cells was used. First, the cells were subcultured and a cell suspension was obtained and thoroughly mixed as to distribute 7

MATERIALS AND METHODS

the cells evenly throughout the suspension. A miniscule sample approximately 1 mL was procured and mixed with approximately 20 L of Trypan Blue. It is important to note that not all cells in the cell suspension are alive, and we must be able to discern the dead cells from those that are still alive. Trypan Blue stains dead cells hence, when counting the cells, we avoid the blue ones. The mixture of suspension and Trypan Blue was placed in the cell counting apparatus, and the apparatus was placed under a microscope. The apparatus is comprised of four 4 4 squares, arranged in a larger 1212 square. The arrangement is such that for any distribution of cells throughout the apparatus, if we were to count the number of cells in the four 44 squares, then the number we obtain will be quite accurate (once we adjust for scaling, which is by a factor of 5000). Once a cell count was obtained, a dilution is necessary to apportion the cells among the wells precisely. Each well can contain 100 L of cell suspension (not accounting for the 100 L needed for the drug solution), and there are 96 wells, which requires 19.2 mL of solution for the entire plate. Depending on the number of plates required, the total amount of solution is subject to variability. The plates were prepared such that each well had 10,000 cells in 100 L, using the formula M1 V1 = M2 V2 . Once the experimental cell suspension was prepared, the experiment could begin. Phase 3. Cell Plate Preparation Each well was lled with 100 L of cell suspension. After one day of incubation (in order to allow the cells to attach the bottoms of the wells), drug application was performed. According the isobolographic method, we must test various concentrations of Cis and Nos in order to determine their IC50 values and hence can were combined with Cis and applied to the cancer cells. Note that the plates were 12 8 plates (hence, 96well plates). The wells were divided into 61 blocks, each block either containing one type of drug combination or a control. Overall, over 12 experiments were done in order to gain enough data to determine the IC50 values of the drugs either alone or in combination. Once the cells had a day to attach themselves to the bottoms of the wells in the plate, the drug solutions had to be prepared. Depending on what concentration was desired, the formula M1 V1 = M2 V2 had to be used along with the molar masses of Nos (413.421
g mol )

and Cis (300.045

g mol ).

After the dilutions were made, 100L

of drug solution was added to each well. The cell plate was labeled and the cells were kept in incubation for 72 hours in order to allow interaction between the cells and drugs. Phase 4. Plate Reading After the 72 hour period of incubation, it was necessary to analyze the amount of living cells remaining so we can see the eects of the drugs upon the cells. This is done by method of absorption spectrophotometry. However, it is necessary to prepare the cells for this process. First, the plates were removed from incubation and the old medium was thrown away using a multichannel pippete. Then, the cells were washed twice with 200L per well of PBS (Phosphate Buer Serum). PBS acts as a cleansing agent it washes away any extra medium or other contaminents. 8

MATERIALS AND METHODS

The process of preparation for the cell reading involves much exposure to mechanical and chemical forces thus, it is necessary that the cells remain rigidly in their place so that the absorption results are not skewed by cells that may have been displaced or killed. A cell xing agent .25% glutaraldehyde keeps the cells rigidly in their place, so that it is easier to read them. 100L of .25% glutaraldehyde was added to each well and the plates were incubated for 30 minutes in order to allow the chemical to take its eect. After the incubation period, the glutaraldehyde was removed and the cells were allowed to dry for 10 minutes. The next chemical to be used .1% crystal violet attaches itself to living cells much like Trypan Blue. However, crystal violet is more accurate in that it can be observed and quantied through an absorption spectrophotometer. Hence, a well which was exposed to a high concentration of Nos or Cis would have a much smaller absorption value than the control would and through this, we can determine the survival rate of the cells and ultimately the IC50 value. Thus, 100L of .1% crystal violet was added to each well. The cells were left at room temperature for 15 minutes, and afterwards the crystal violet was removed. Crystal Violet is a very strong substance and will easily attach to dead cells or the walls of the plate, hence, it was necessary to vigorously wash the plates in distilled water until all violet was removed except for any crystal violet attached to living cells. Note that this is possible without harming the cells because we exposed them to .25% glutaraldehyde, the cell xing agent. Finally, when the plates were dry after the washing, 100L of a substance called sodium dihydrogen phosphate was added to each well. Sodium Dihydrogen Phosphate acts as a buer and is necessary in order to perform the absorption process. Finally, the cells were read. Using a Quant absorption spectrophotometer, the plates were placed in and read at a 540 nm wavelength. After the machine was nished, an Excel sheet of absorption values was generated by the machine and the percent survival rates could be computed. The cells and plates were disposed of accordingly after bleaching. The experiment concluded.

RESULTS

Results
The ultimate goal of the experiment was to determine the combination index values of Noscapine and

Cisplatin. The combination indices quantify the strength of the relationship between the two drugs against the H460 and A540 cell lines. In order to determine the combination index, we must know the IC50 values of the two drugs. The drugs were tested at various combinations and the percent rate of death was computed. The results from the combination studies were averaged and compared to the averages from the studies of Noscapine and Cisplatin alone; the results are shown below in the The IC50 value is a measure of the eectiveness of a compound in inhibiting the biological function of an antagonist agent. Contextually, this is the concentration of the drug necessary to deplete 50% of the cancer cells. To determine this value, we must determine the percent survival rate of the cancer cells in the cell plate study at various concentrations of the drug. Once this is done, we can plot percent survival rate against concentration and plot a best t line and then use interpolation to determine the concentration when the percent survival is rate is 50%. For example, in one experiment, we tested Noscapine at various concentrations from 20320M and used interpolation to determine Noscapines IC50 value for the H460 cell lines. The graph is shown below.

The drugs were tested at various combinations and the percent rate of death was computed. The results from the combination studies were averaged and compared to the averages from the studies of Noscapine and Cisplatin alone; the results are shown below in Figure 2.

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RESULTS

The Noscapine and Cisplatin combination treatment had an average percent kill rate of 78.1 7.5% compared to 38.2 6.8% by Cis and 35.4 6.9% Nos alone. Hence, it is clear that there is an additive eect between the two. However, simply looking at the percent killed is not enough to generalize a synergistic relationship. The percent killed is somewhat blinding; it is not enough to generalize over many concentrations. Thus, the IC50 value is chosen as the base indicator of a drugs strength, and it is what we shall use to analyze the drugs. The IC50 values obtained are shown in the table below. Table 1. IC50 Values (M) of Noscapine and Cisplatin for H460 and A549 NSCLC Cell Lines H460 Noscapine 34.7 Cisplatin 1.4 A549 Noscapine 61.25 Cisplatin 4.2

The IC50 values are necessary to determine the combination indices of the drugs. The combination index (CI) is determined by the following formula: CI = D1 D2 D1 D2 + + Dx1 Dx2 Dx1 Dx2

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4 where Dx1 = Dose of drug 1 to produce 50% cell kill alone; D1 = Dose of drug 1 to produce 50% cell kill in combination with D2 ; Dx2 = Dose of drug 2 to produce 50% cell kill alone; D2 = Dose of drug 2 to produce 50% cell kill in combination with D1 ;

RESULTS

= 0 for mutually exclusive mechanisms or 1 for mutually nonexclusive mechanisms (in this case, = 0) The combination indices were tested at the IC50 value of Cisplatin and various concentrations of Noscapine, namely, 10, 15, 20, 30, 40, and 50 M. This was done to see the aect of dierent concentrations of Noscapine upon a literature concentration of Cisplatin, this constancy of the IC50 value of Cisplatin acts as somewhat of a control and allows analysis to be much easier. The combination indices are shown in Table 2.

Note the following ratings for combination indices: CI > 1.3 : antagonism 1.1 < CI 1.3 : moderate antagonism 0.9 < CI 1.1 : additive eect 0.8 < CI 0.9 : slight synergism 0.6 < CI 0.8 : moderate synergism 0.4 < CI 0.6 : synergism 0.2 < CI 0.4 : strong synergism

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DISCUSSION AND CONCLUSION

Discussion and Conclusion

As illustrated by the results above, it was found for all concentrations of Noscapine used, the combination

index indicated at least synergism if not strong synergism. The lower combination values indicate that a lower dosage of the drug when used in combination with the secondary drug is necessary for 50% rather than when used alone. Hence, the ratio of the dosages of the drugs is closer to 0, indicating a smaller CI and furthermore, synergism. The IC50 values of Noscapine obtained are consistent with literature values of IC50 values for other cancers. Though this study is novel in that it is the rst to test Noscapine against H460 and A549 lung cancer cell lines, we can expect them to be comparable to the cytotoxic eects observed with murine melanoma B16LS9 cells (IC50 = 50M) and human ovarian 1A9PTX10 cells (IC50 = 22.7M). Thus, we can trust the IC50 values of Nos obtained from the study. Overall, we observed CI values ranging from 0.31 0.5 to 0.57 0.4 for 50% cell kill; this range clearly indicates the synergistic to strongly syngergistic relationship. While this agrees with our hypothesis, we must take in to account the fact that there may be error. For one, interpolation is not an errorfree process determining the IC50 value through use of a graph can often err, and the only way we can trust our answer is through the addition of more concentrations tested in order to establish a more accurate best t curve. Furthermore, the cell counting process is not fully accurate and is loosely based on overall trends in the distribution of cells. While in the long run the error may be limited, we must still account this for any possible error. Despite this, the nal errors 0.5 and 0.4 are small enough that we can trust the results as accurate throughout the entire experiment. The isobolographic method is desirable in that it safely generalizes the behavior of various concentrations of Noscapine using the IC50 value and the combination index the need for graphs of percent kill vs. concentration for concentrations ranging through the hundreds is unwanted and tedious. The combination index provides a concise and accurate quantity that describes the relationship between the drugs in a pleasing manner and allows us to establish a generalization without becoming lost in a sea of graphs. The implications of this study are tremendous. It is novel in that it is the rst study to examine the relationship between Noscapine and Cisplatin on the lung cancer cell lines H460 and A549. Hence, with the desirable results obtained, that is, the massive increase in the ecacy of Cisplatin due to Noscapine, it gives hope that a new treatment may arise that may limit the cytotoxicity of Cisplatin. However, this experiment is only the foundation of a series of studies necessary in order to fully understand the relationship between the two drugs. This study is strictly in vitro; an in vivo study, possibly using lung tumors in mice may help give insights into the nature of the synergistic relationship between the drugs in a real life situation. Furthermore, the connection between the two drugs mechanisms is unknown; in order to examine

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DISCUSSION AND CONCLUSION

how they interact with the apoptotic pathway of a lung cancer cell line, Western Blots must be performed on cell lysates so the underlying proteins involved can be analyzed. Nonetheless, this experiment is only the start of a possible novel treatment involving Noscapine and Cisplatin. With further research, it may be possible in the new future to rene chemotherapy using Noscapine such that it is more ecacious and secure. Thus, from experimentation, we can conclude that from both the percent death rate and the combination indices, Noscapine and Cisplatin share a synergistic behavior when exposed to the lung cancer cell lines H460 and A549. This nding agrees with our initial hypothesis and opens the door to further experiments and ndings in the eld of chemotherapy and Human NonSmall Cell Lung Cancer.

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BIBLIOGRAPHY

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