1. Apoptotic Activity of Diallyl Trisulfide in Cancer Cells
Results
Introduction
Cells- OPM2 cells were cultured in recommended media and used at passages Cultures were maintained at 37°C in a humidified atmosphere of 5% CO2 and 95% air.
Cell viability- Cells were plated in a 96-well plate at a density of 40k cells/well in triplicate. Cells were treated with varying conc. of DATS (shown in Figure 2) for 24h. The viability was assessed by MTS assay and the absorbance was measured at 490nm wavelength.
Microscopy- Cells treated with different conc. of DATS (Figure 1) for 24h were observed under a microscope.
Flow Cytometry- Cells were plated at a density of 0.3M cells/well in a 24-well plate and treated with different doses of DATS (shown in Figure 3 & 4) for 24h. Cells from each conc. were stained with FITC-conjugated anti-CD138 antibody, APC-conjugated Annexin V, and Propidium Iodide (PI) for 20 min. After adding binding buffer, cells were analyzed with the FACS Caliber flow cytometer, using CellQuest software (BD Biosciences) and FCS Express V3 (De Novo Software).
Western Blot- After preparing protein lysates, 15µg of protein were resolved on NuPAGE Novex Bis-Tris mini gels and transferred to PVDF membranes. Membranes were probed with the antibodies caspase-3, cleaved caspase-3, and GAPDH. The next day, blots were exposed to HRP- conjugated secondary Ab for 1 hour and detected by chemiluminescence.
Conclusions
Materials & Methods
Diseases involving abnormal and rapid cell proliferation, potentially able to metastasize, are classified as cancerous. About 1.7 million Americans were diagnosed with a form of cancer this past year. A malignancy of plasma cells, multiple myeloma (MM) accounts for about 13% of all hematological cancers. MM results in a weakening of the immune system, reduction in RBC and hemoglobin production, and weakening of bones. MM is incurable; approximately 46% of patients diagnosed with MM die from it annually.
In the past decade, active agents, like thalidomide or bortezomib, and therapies, such as hematopoietic stem cell transplants or chemotherapy, have emerged as possible treatments for myeloma. These existing cancer drugs and treatments all rely heavily on the induction of apoptosis to kill cancerous cells.
Allium sativum, or garlic, has various medicinal properties and has been used for centuries to treat common ailments. Recent studies have provided evidence that garlic reduces the risk of heart disease, due to its increased levels of hydrogen sulfide, as well as providing cardiovascular support and preventing degenerative conditions like atherosclerosis. Allicin, the active ingredient of garlic, produces diallyl trisulfide (DATS) when decomposed. This potent organosulfur has been attributed to many of the health benefits of garlic. Studies have shown that DATS aggregates platelets, reduces blood pressure, and has the ability to selectively kill cancer cells in the prostate and breast.
The present study investigates the treatment of the MM cell line OPM2 with DATS in order to evaluate its antiproliferative effects.
DATS inhibits growth of OPM2 myeloma cells in a dose-dependent manner as suggested by MTS assay
OPM2 cell viability is suppressed through induction of apoptosis; flow cytometry analysis indicated that as DATS concentration increased, cells staining positive for propidium iodide (PI) and Annexin V increased drastically
Apoptosis induced by DATS occurs through a caspase-dependent pathway, as shown by western blot analyses
Ability of DATS to induce apoptosis in MM cells indicates potential promise as cancer preventive agent
Further Research- Further study into the properties of DATS is still necessary for the elucidation of its inhibitory effects on MM. Additional western blotting could be performed to analyze the apoptotic pathway DATS induces in MM cells more specifically, i.e. intrinsic or extrinsic. Furthermore, DATS could be combined with existing chemopreventive drugs to examine potential synergistic effects. It would also be interesting to see if DATS has similar effects on related hematological cancers, such as leukemia.
Apoptotic Activity of Diallyl Trisulfide in Cancer Cells
OPM2 cells after treatment with DATS (24h)
Cell Viability after 24h exposure to DATS
(MTS assay) A B C D E F G H I J K L
Observations & Discussion
Morphological Changes-
Microscopic images of OPM2 cells, shown in Figure 1, depict the continuous degradation of cells treated with increasing concentrations of DATS. In 15.6µM and 32.5µM, a few cells had visible morphological alterations, but in 62.5µM and 125µM, less than half of the cells were still healthy and unharmed. It can be noted that in 250µM, very few cells remained viable; most had been reduced to apoptotic bodies.
Cell Viability-
Displayed in Figure 2, the MTS assay showed that cell proliferation decreased exponentially in a dose-dependent manner when treated with 1, 2, 4, 8, 16, 32, 64, and 128µM of DATS for 24 hours. An ANOVA statistical analyses was conducted on MTS data. The data was shown to be highly significant, with p<
Flow Cytometry-
Flow cytometry was conducted to study DATS-mediated triggering of apoptosis. One of the earliest features of apoptosis is the degradation of the cellular membrane. The marker Annexin V can identify early apoptotic cells as it binds to cells undergoing membrane degradation. It was used in conjunction with Propidium Iodide (PI), a fluorochrome that stains for dead cells. Figure 3 shows dot plots, with PI plotted against Annexin V APC, at increasing concentrations of DATS.
In the control plot, almost 90% of cells tested negative for both markers, meaning these cells were viable. 3.1, 6.2, and 12.5μM were similar. However, from 12.5μM to 25μM, the amount of cells testing positive for both markers shot up by 30%. In 25μM, there was also a large cluster of cells testing negative for Annexin V but positive for PI, signifying late apoptosis. In 50μM and 100μM, 90-95% of cells were split between the top two quadrants, both positive for PI, meaning the majority of cells had fully undergone apoptosis at these concentrations.
Western Blot-
DATS-induced apoptosis was markedly elevated in OPM2 cells, as shown by cleaved product of caspase-3 (Figure 6).
Fig 1: DATS induced morphological changes in OPM2 cells
Fig 2: DATS rapidly decreases OPM2 cell viability in a dose-dependent manner
Apoptotic Activity in OPM2 cells After 24h Exposure to DATS (Flow Cytometry)
Control
3.1μM
6.2μM
12.5μM
25μM
50μM
100μM
Fig 3 & 4: Flow cytometry indicates that OPM2 cells undergo apoptosis when treated with DATS
Fig 5: Apoptotic Cycle
Source:
2.5 5 10 20
DATS (μM) 40
Caspase-3
35kDa
19kDa
17kDa
GAPDH
37kDa
Cleaved
Western Blot Analysis
(After 24h Treatment)
Fig 6: DATS-mediated apoptosis in OPM2 cells is activated via a caspase-dependent pathway