Ranging from herbs to acupuncture, alternative medicine is becoming increasingly popular for managing health-related issues. In this brief report, we described that frankincense oil, with a window of concentration, specifically suppressed cell viability in human bladder carcinoma J82 cells, but did not affect cell viability in immortalized normal urothelial UROtsa cells. Frankincense oil suppressed J82 cell viability may be attributed to the activation of growth arrest and pro-apoptotic genes. The possibility that the witnessed differences in cell survival could be due to the presence of EGF in the media was considered. Despite this concern, we expected that UROtsa cells' resistance to frankincense oil as compared to J82 cells may not result from the presence of EGF in their growth medium based on the following observations: first, a higher concentrations of EGF (i.e. 1 μg/ml) had been reported for apoptosis protection [27
], second, UROtsa cells cultured in EGF-free medium were less sensitivity to frankincense oil-suppressed cell viability as compared to J82 cells, although the overall UROtsa cell viability was reduced (data not shown), and third, four other oils, including sandalwood oil (Santalum album
), balsam fir oil (Abies balsamea
), palo santo oil (Bursera graveolens
), and tsuga oil (Tsuga canadensis
) (Young Living Essential Oils), induced nearly identical cytotoxicity in both J82 and UROtsa cells (data not shown).
Commercial frankincense oil was directly applied in our experimental cell models without modification. It was not our intention in this preliminary study to dissect the specific chemical composition of frankincense oil nor determine its efficacious dosage, since some reports indicated that total frankincense extract is more potent than pure, specific boswellic acids [9
]. However, a standard assessment between chemical composition of frankincense oil and its efficacy in tumor suppression will be required in future clinical trials. In addition, frankincense oil was directed added to cell culture media in this study without the inclusion of a carrier; and dose-dependent suppression in cell viability was observed in both J82 and UROtsa cells in the absence of any oil carrier. The absence of carrier eliminated carrier-dependent effects of frankincense resin extract as reported by Chevrier et al
Gene expression analysis was terminated within 3 hours following frankincense oil treatment, since isolated RNA quality and quantity were not sufficient for microarray analysis beyond this time point. We reported a total of 122 up- and 47 down-regulated genes with greater than 2-fold induction or suppression over the period of 3 hours. These findings suggested very specific actions of frankincense oil. The genome-wide gene expression analysis supports patterns of stress, activation of cell cycle arrest, suppression of cell proliferation, and activation of apoptotic signaling in frankincense oil-treated J82 cells within 1 hours of stimulation, and some of these processes were sustained for 3 hours.
Based on the temporal regulation of the genes identified by microarray and bioinformatics analysis, we proposed that frankincense oil induces various death pathways in J82 cells. Waves of transcription factors were regulated by frankincense oil from between 30 min and 3 hours. EGR1 was one of the few genes that were rapidly up-regulated within the first 30 min. Although EGR1 has been shown to be an early gene that is immediately up-regulated in other systems and is correlated with DNA repair [28
], the mechanism for elevated EGR1 expression by frankincense oil is unclear. EGR1 has been reported to increase transcription of another transcription factor ATF3 [29
]; and levels of ATF3 were increased in our system between 30 and 60 min after frankincense exposure. ATF3 is induced by stresses and can bind to DDIT3 [30
], which was up-regulated between 2 and 3 hours in our system. In addition, DDIT3 responds to DNA damage [31
], and is responsible for cell cycle arrest [32
]. Moreover, using the PAINT webtool http://www.dbi.tju.edu/dbi/tools/paint/
] to search the TRANSFAC database, we identified EGR1 and ATF3 binding sequences upstream of multiple apoptosis-related genes identified in our system. EGR1 can bind 5'-flanking regions of 5 of the identified apoptosis-related genes (FGFR1, GADD45B, HES1, RHOB, and TRIB1) that were up-regulated between 2 and 3 hours. Binding sites for ATF3 were found in the 5'-flanking regions of 3 apoptosis-related genes (FOSB, GEM, and LAMA5) that were up-regulated between 1 and 2 hours, and 3 additional genes (HSPA1A, ID1, and JUN) between 2 and 3 hours. By similar inference, ATF3 may also account for the expression of 2 down-regulated genes: ING4 (1–2 hours) and ATG5 (2–3 hours). The sequential expression of these identified transcription factors may be ultimately responsible for cell cycle arrest, suppressed cell proliferation, and apoptosis in frankincense oil-treated J82 cells.
Suppressed cell viability and proliferation in frankincense oil-treated J82 cells was also confirmed by elevated expression of genes that are responsible for cell cycle arrest and anti-proliferation. Up-regulated IL8 [34
] and CDKN1A [35
] have been shown to be responsible for cell cycle arrest and suppressed cell proliferation. IL1A is a negative regulator for cell cycle progression and cell proliferation [36
]; and IL6 has been shown to induce growth arrest [37
]. However, levels of ING4, a molecule that is a negative regulation of cell proliferation in a human hepatocellular liver carcinoma cell line (HepG2) [38
], were up-regulated in frankincense oil-treated cells. ING4 may function differently between J82 cells and HepG2 cells; or ING4 activity is suppressed by a large number of pro-apoptotic molecules in response to frankincense oil.
Frankincense oil up-regulated several pro-apoptotic genes, including CDKN1A [39
], DEDD2 [40
], NUDT2 [41
], SGK, TNFAIP3, and IER3 [42
]. Elevated expression (between 0.5 and 2 hours) followed by suppressed expression (2–3 hours) of a cell survival membrane receptor, AXL [43
], suggests that cells may try to prolong cell survival following frankincense oil exposure. We also propose that frankincense oil might activate both extrinsic and intrinsic death signaling in J82 cells through stress and death receptors, respectively, to execute apoptosis. Intrinsic death signaling was suggested by up-regulated expression of SSRT1, GADD45B, DDIT3, CDKN1A that have been shown to be required for DNA damage-induced cell cycle arrest [35
]. Extrinsic death signaling was implicated by the up-regulated expression of DEDD2, which is a death domain receptors and induces apoptosis [40
Although the bioinformatics and TUNEL analyses reported here suggested that frankincense oil induced apoptosis, rather than necrosis, in J82 cells, frankincense oil did not cause DNA fragmentation, a hallmark of apoptosis, in this bladder cancer cell line. It is possible that DNA fragmentation occurred between 6 and 12 hours post frankincense oil treatment. Alternatively, apoptosis without DNA fragmentation has been reported in several occasions [44
]; and frankincense oil-induced J82 cell death may fit in this category. The detailed molecular and biological pathways utilized by frankincense oil in inducing bladder cancer cell specific cell death require further investigation.
This study helps to show that frankincense oil may be appropriate as an alternative therapy for bladder cancer. This is the first report demonstrating that frankincense oil can discriminate between bladder cancer cells and normal urothelial cells in a cell culture system, and utilizing microarray technology to identify potential biological pathways activated by frankincense oil. Our results are consistent with a news report that frankincense oil specifically targets malignant melanoma but not normal skin cells in horses http://www.purepeace.com/press/press_frankincense.pdf
. Future studies are required to determine whether frankincense oil has similar effects on other bladder cancer cell lines of varying severity such as RT4, T24, and 5637, followed by in vivo
studies using bladder cancer animal models. In addition, a standard manufacturing and indication needs to be applied before the commercial frankincense oil can be used as an alternative or complementary therapy for treating bladder cancer.