By Abinav Sankaranthi and Arnav Sankaranthi, March 2018.
Introduction
According to the World Health Organization, globally, 1 in 6 deaths are due to cancer.
70% of the deaths from cancer occur in low and middle-income countries [7]. Lung cancer is the world’s leading cancer killer.
There are three main types of lung cancer namely non-small cell lung cancer (most common), small cell lung cancer and lung carcinoid tumor. Treatment Options include surgery, radiation therapy, chemotherapy. Even though the FDA has approved certain drugs that work against many human cancers, cancer is still an incurable disease. The existing therapeutic applications fail because of drug resistance, side-effects, and recurrence of cancer. Current cancer treatments that involve chemotherapy impact non-targeted tissues, creating adverse side-effects in humans. Therefore, therapeutic agents that only target the cancer cells without harming normal cells are needed. In this regard, plant-based alternative treatments and therapies are gaining attention as and when they are identified safe and non-toxic to normal cells.
For centuries, plant-based medicines have been used in developing countries like India. Anticancer agents such as curcumin, green tea extract, and so on were identified by many researchers from natural sources [6]. Our research focused on using plant-based repurposed medicines that are available to low and middle-income countries for the prevention and treatment of one of the most common cancer -- lung cancer. Furthermore, we established a cancer cell culturing and repurposed drug screening model in a high school lab.
Drug repurposing is an application of well-characterized drugs or compounds to new diseases. This cuts both the costs and the time in bringing new drugs especially those that cater to the many diseases neglected with economics in the mind of the big pharmaceutical corporations. We chose PVL14, a natural plant-based medicine used in Ayurveda as a low-cost repurposed drug to screen for anti-cancer activity. This plant extract contains triterpenoids that are potentially promising chemo-preventive agents [5]. Triterpenoids are hydrocarbons composed of three terpene units. Triterpenes like lupane, oleanane, and ursane have shown pharmacological potential for cancer treatment. In our research, we used human lung adenocarcinoma A549 cell cultures to study the effect of the PVL14 drug on cancer cell proliferation. We hypothesized that if different concentrations of PVL14, a plant-derived compound, are applied to A549 lung cancer cells, then we will see a reduction in the proliferation of the cancer cells.
Our approach established a 2D cell culture and drug treatment model in our high school lab to screen plant-derived compounds and study their anti-cancer effects. In the first stage of the three-stage process, A549 human lung cancer cells were grown and maintained in culture flasks containing growth medium with antibiotics. Cells were passaged as they reached high confluency. In the second stage, A549 cells were harvested at equal density in multiple 12-well plates. A stock solution of PVL14 dissolved in ethanol and serially diluted with growth medium was made, to obtain a range of low, medium, and high concentrations of the drug solution. After
overnight proliferation, each of the wells was treated with the corresponding drug solution. The control group was untreated A549 cells harvested in the growth medium. All treatments were performed in triplicates. Nutrient supply and temperature were the controlled factors. The final stage involved counting live cells to determine cell proliferation at 24-hour, 48-hour, and
72-hour time intervals.
Materials and Methods
Materials
The PVL14 drug was obtained from PACT Health LLC (Connecticut, USA). PVL14 is a repurposed drug originally used for treating osteoporosis. The exact composition of this
plant-derived extract was not revealed by PACT Health LLC. In this study, the concentration was expressed as the amount of PVL14 per milliliter of media holding the cells (mg/mL).
The A549 lung cancer cells were obtained from Sigma-Aldrich. The Gibco lung cancer kit was purchased from Thermo Fisher and it contained basal media, fetal bovine serum, penicillin-streptomycin, trypLE express enzyme. opti-MEM I, Nunc cell culture treated flasks. The Trypan Blue solution (0.4% liquid sterile and filtered) was obtained from Sigma-Aldrich.
Lab equipment used included a Moxi Flow Cytometer, Pipettes, Culture Flasks, Centrifuge tubes, Incubator, Freezer, BioSafety Hood and Protective Equipment, Inverted Microscope, Timer and Counter, MotiCam image capturing software, and Graphing Software such as Excel. All equipments used were from Valley Christian High School science research lab.
Procedure
Based on prior research drug treatment studies conducted with green tea extract, a 2D cell culture and drug treatment model to screen plant-derived compounds and study their anti-cancer effects was established[1]. The procedure involved three stages. In the first stage, the A549 human lung cancer cells were grown and maintained in culture flasks containing growth medium with antibiotics. Following the cell culturing and cell passage methods described in section 2.3, the A549 cells were passaged as they reached high confluency. In the second stage, the A549 cells were harvested at an equal density of 1.0 X 10^4 cells/well in multiple 12-well plates.
Following the serial dilution and treatment method described in section 2.6, a stock solution of PVL14 dissolved in ethanol and serially diluted with growth medium was made, to obtain a drug concentrations ranging from 0.3125 mg/mL to 5 mg/mL. After overnight proliferation, each of the wells was treated with the corresponding drug solution. The control group was untreated A549 cells harvested in the growth medium. All treatments were performed in triplicates.
Nutrient supply and temperature were the controlled factors. The final stage involved counting live cells to determine cell proliferation at 24-hours, 48-hours, and 72-hours time intervals.
Cell Culturing and Passaging Method
To prepare the initial cell culture, the vial with cancer cells was thawed in a 37 degrees Celsius water bath for 1 to 2 minutes. Ethanol was sprayed outside of the cell vial and was then moved to the hood. Then, 19 mL of complete media was added along with 1 mL of thawed cells to the T-75 flasks. The flasks were gently moved in the direction of north/south and east/west for mixing. Flask was then labeled and incubated at 37 degrees Celsius for a few days. When the flask reached 80% confluency, passaging procedure was started. The cell counting method described in section 2.5 was followed to count the number of live cells per milliliter.
Cell Plating Method
From the cell count number, the volume of cells in the basal media per well was calculated to obtain a 1 x 10^4 cells/mL initial density for each well. In 12-well plates, growing A549 cells were harvested and seeded at an initial density of 1 x 10^4 cells/mL in 12-well plates. Each well was clearly labeled with time period and drug dilution concentration. Plates were placed in the incubator at 37 degrees Celsius and left for overnight proliferation.
Cell Counting Method
To count the cells, old media was aspirated out. A 0.4 mL of PBS was added to each well and then aspirated out. Then a 0.3 mL of Trypsin-EDTA was added to each well. The well plates were placed in the incubator at 37 degrees Celsius for 5 minutes until all the cells were detached. Then a 0.6 mL of complete media was added and pipetted up and down to make a single suspension, free of many clumps. From this, 15 microliters of cells were taken from each well and placed in a PCR tube. Then, 135 microliters of viability reagent were taken into the PCR tube and counted using Moxi Cassettes (70 microliters per test).
Serial Drug Dilution Method and Treatment
To obtain the necessary concentrations of the PVL14 drug for treatment, serial dilution method was used. 100 mg of PVL14 was taken and added to 0.4 ml of Ethanol. The mixture was vortexed and let stand for 15 minutes. 6 empty tubes were taken and marked 1 through 6 for serial dilution. From the vortexed and settled drug solution, 0.32 mL of the supernatant was taken and added to the tube 1. To this tube, 15.68 mL of growth medium was added to make a total of 16 mL, resulting in a drug concentration of 5 mg/mL in tube 1. The solution in the tube 1 was gently mixed by closing the tube and rotating and pipetting up and down. Serial dilution was started by taking 8 mL from the tube 1 and adding it to the tube 2. Then 8 mL of growth medium was added to the tube 2 to make a total of 16 mL, resulting in a drug concentration of 2.5 mg/mL. The solution in the tube 2 was gently mixed by closing the tube and rotating and pipetting up and down. Serial dilution was followed for the rest of the tubes resulting in 1.25 mg/mL, 0.625 mg/mL, and 0.3125 mg/mL. In the last tube marked 6, a 0.16 mL of ethanol and a 7.84 mL of growth medium was added to make a total of 8 mL. This tube was used for the control group. After overnight proliferation, growing A549 cell plates were pulled out from the incubator. From each of the cells in the well plates, growth medium was aspirated out. From the corresponding drug solution tubes, 0.75 mL of the drug solution was added to each of the wells in triplicates of drug concentration. Once treatment is done, for every set (set A, set B, and set C corresponding to 24-hour, 48-hour and 72-hour wells) we had triplicates of each of the concentrations of the drug and 1 triplicate for the control group. These plates were placed in the incubator at 37 degrees Celsius. Using the cell counting method described in section 2.5, cells were counted in respective well plates after 24 hours, 48 hours, and 72 hours of drug treatment.
Results and Discussion
The results of our experiments showed a decrease in cell proliferation count from the plating density after 24-hours, for a higher dose of the drug specifically above 2.5 mg/mL. At the 48-hour time interval after drug treatment, a decrease in cell proliferation for all dosages was seen except at 0.625 and 1.25 mg of the drug. At the 72-hour mark, the lower concentrations had the most effect in reducing cell proliferation, whereas at higher concentrations of drug above
1.25 mg/mL, the live cell count had increased. Between 48 hours and 72 hours at a higher dose of the drug, we saw an increase in the cell proliferation. This may be due to the depleted drug in the wells. The results showed modulations in the proliferation of cancer cells both with drug dosage and over time. Further investigative studies are needed to study this effect more deeply.
Figures and Tables
Figure 1
A549 cell growth in media
(A) A549 cell proliferation under a microscope. (B) Zoomed in view of the more than 80% confluent A549 lung cancer cells
Figure 2
Effect of PVL14 on A549 live cell count at 24 hours for PVL14 concentrations at (0, 0.3125, 0.625, 1.25, 2.5, 5 mg/mL)
Figure 3
Effect of PVL14 on A549 live cell count at 48 hours for PVL14 concentrations at (0, 0.3125, 0.625, 1.25, 2.5, 5 mg/mL)
Figure 4
Effect of PVL14 on A549 live cell count at 72 hours for PVL14 concentrations at (0, 0.3125, 0.625, 1.25, 2.5, 5 mg/mL)
Figure 5
Microscope view of cell plates at 24-hour (A), 48-hour (B), and 72-hour (C) after drug treatment for control group (0mg/mL) and treated cells groups (0.3125, 0.625, 1.25, 2.5, 5 mg/mL)
Table 1
Number of Live and Dead (non-floating) Cells with various concentrations of drug over 24 hours, 48 hours and 72 hours after drug treatment.
Conclusion
The purpose of our research was to screen for anti-tumor properties of plant extracts that are currently used to treat non-cancer conditions. The PVL14 is one such plant-derived drug that is used for treating osteoporosis. This study investigates the effect of the repurposed plant-derived extract, PVL14 on the proliferation of A549 lung cancer cells. The PVL14 plant-derived drug appears to have an effect on the proliferation of A549 human lung cancer cells based on our preliminary investigative study. For the various concentrations of the drug, we saw modulations in the cancer cell proliferation rate. For lower concentrations of the drug, the live cell count decreased over time. Whereas for higher concentrations of the drug, we saw a profound decrease of cell proliferation at 48 hours and an increase at 72 hours. Because of the modulations in the proliferation cell count with various concentrations of the drug, it is inconclusive as to the effectiveness of the PVL14 plant extract on reducing the A549 cancer cell proliferation at the tested concentrations and time intervals. For higher dosage of the drug, we need to do further investigation to study if a rapid depletion of the drug by 48 hours initiated an increase in cell proliferation by 72 hours. In contrast to the study conducted on the effect of green tea extract on A549 lung cancer cells, where proliferation rate decreased as GTE concentrations increased, the data from PVL14 experiments show modulations on the proliferation rate. These effects could have also occurred due to limitations in the methods used such as calibration inaccuracies in the equipment as well as cell clumping. We will also continue to run experiments to investigate if a repetition of PVL14 drug dosage at various time intervals has any impact on the A549 cancer cell proliferation. The studies imply that there are opportunities to identify plant-based drugs as alternatives for cancer treatment.
Future Research
Future research includes investigating if the second dosage of drug treatment at 48 hours for higher concentrations of PVL14 reduces cell proliferation. This study could be expanded to establish 3D cell culture model for A549 cancer cells and study the effect of PVL14 using 3D models. Further studies could screen other plant-based compounds to study anti-cancer cell activity on A549 lung cancer cells and other cancer cell types. A comparison study of the effect of plant-based and non plant-based drugs on the cancer cell proliferation and apoptosis could be performed in the future.
Acknowledgments
This study has been supported by Valley Christian High School covering all the materials and lab use costs. We are grateful to Mr. Vanderveen, co-chair of Valley Christian High School Science Department and Dr. Papineni, CEO of PACT Health and adjunct faculty at KUMC for providing guidance and support throughout our study.
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