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Holy Basil leaf extract decreases tumorigenicity and metastasis of aggressive human pancreatic cancer cells in vitro and in vivo: Potential role in therapy
Source:
Shimizu T, Torres MP, Chakraborty S, et al. Cancer Letters. Volume 336, Issue 2, 19 August 2013, Pages 270–280

There is an urgent need to develop alternative therapies against lethal pancreatic cancer (PC). Ocimum sanctum (“Holy Basil”) has been used for thousands of years in traditional Indian medicine, but its anti-tumorigenic effect remains largely unexplored. Here, we show that extracts of O. sanctum leaves inhibit the proliferation, migration, invasion, and induce apoptosis of PC cells in vitro. Isolates include methyl eugenol, the most abundant, with eugenol, linalool, apigenin, orientin and vicenin 
The expression of genes that promote the proliferation, migration and invasion of PC cells including activated ERK-1/2, FAK, and p65 (subunit of NF-κB), was downregulated in PC cells after O. sanctum treatment. Intraperitoneal injections of the aqueous extract significantly inhibited the growth of orthotopically transplanted PC cells in vivo (p < 0.05). Genes that inhibit metastasis (E-cadherin) and induce apoptosis (BAD) were significantly upregulated in tumors isolated from mice treated with O. sanctum extracts, while genes that promote survival (Bcl-2 and Bcl-xL) and chemo/radiation resistance (AURKA, Chk1 and Survivin) were downregulated. 
Overall, our study suggests that leaves of O. sanctum could be a potential source of novel anticancer compounds in the future.
Discussion
O. sanctum, also known as Ocimum tenifluorum or Holy Basil, an herb belonging to the family Lamiaceae, is a well-known medicinal plant in use for several thousands of years in the traditional Indian system of medicine. While most studies have focused on the therapeutic effects of O. sanctum leaf extracts on benign diseases, studies of its anti-cancer effects are sparse.
To our knowledge, there are no published studies of the effect of basil extracts in PC cells. We observed that both EOOS and EEOL significantly inhibited the motility and invasion of PC cells in vitro. This was associated with a significant downregulation of activated FAK, a key mediator of both these processes in PC cells. In vivo, while AEOL significantly decreased tumor size, we did not observe any significant difference in the incidence of macroscopic metastases between animals treated with AEOL or vehicle (water). However, there was a strong upregulation of E-cadherin in the tumors of the AEOL treated mice. 
One possibility for the lack of observable effects on metastasis in vivo is that PC cells establish metastasis very early, when the tumor is still small. Since the mice were treated with AEOL after 7 days post-orthotopic implantation, we hypothesize that micrometastasis had already been established by the time the therapy was started. A comparison of the IC-50 values between AEOL and EEOL revealed that the former was nearly 20–40-fold less effective at inhibiting PC proliferation in vitro. This suggests that a higher AEOL dose in our in vivo experiment may have inhibited the proliferation of metastatic deposits. Our future studies will aim to determine the effect of individual compounds present in EOOS in inhibiting the aggressive behavior of PC cells in vivo.

Additionally, we observed that in vitro incubation of AsPC-1 cells with EOOL and EOOS and in vivo treatment of orthotopically implanted AsPC-1 cells with AEOL downregulated the expression of Survivin, a gene that has been previously shown to be a key mediator of chemo- and radio-resistance in PC and other solid tumors [1-4]. Thus, its downregulation in PC cells suggests the possibility that compounds present in O. sanctum leaves could potentially sensitize PC cells to chemotherapy and radiation. We are currently evaluating the effect of EEOL (of NC) and EOOS on both the sensitization (by pre-treatment) and the efficacy (in combination with) of PC cells to conventional chemotherapy. We are also evaluating the role of major compounds found in basil [5,6] in PC therapeutics. An analysis of essential oil from fresh leaves of O. sanctum identified nearly 16 different constituents [7]. Of these, methyl eugenol is the most abundant, with eugenol, linalool, apigenin, orientin and vicenin being some of the other components.

A limitation of our study is the use of dried vs. fresh basil leaves to prepare the extracts. While this might have resulted in the lower magnitude of effects in cells treated with EEOL/AEOL (compared to EOOS treated cells), it gave us valuable insight into the potential clinical benefits of commercially marketed basil supplements. Fresh basil leaves are difficult to obtain and store in quantities large enough for long-term studies and may show variation from batch to batch. On the other hand, we obtained the dried leaves from four independent vendors and used the same lot for the entire experiment. Given the lack of reliable scientific evidence to support the use of most health supplements, our study, through its unbiased and broad-based experimental strategy, provides the first evidence for the potential utility of basil extract for the treatment of one of the most lethal malignancies known to man.

In conclusion, we report that ethanolic extract and the essential oil of O. sanctum inhibit the proliferation, motility and invasive ability of PC cells. Intraperitonally injected aqueous extracts decreased the tumorigenicity of orthotopically implanted AsPC-1 cells significantly. This was associated with significant increase in the expression of pro-apoptotic genes with a concomitant decrease in the level of genes that inhibit apoptosis or promote PC cell proliferation or metastasis. Taken together, these results suggest that the extract or essential oil of basil leaves and the components present in them could be potentially useful as novel agents for the therapy and/or prevention of human PC. These results will form the basis for future studies to investigate the effect of individual components of O. sanctum leaves for the treatment of PC.

References
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3. A.C. Mita, M.M. Mita, S.T. Nawrocki, F.J. Giles. Survivin: key regulator of mitosis and apoptosis and novel target for cancer therapeutics. Clin. Cancer Res., 14 (2008), pp. 5000–5005
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5. M.A. Kelm, M.G. Nair, G.M. Strasburg, D.L. DeWitt. Antioxidant and cyclooxygenase inhibitory phenolic compounds from Ocimum sanctum Linn Phytomedicine, 7 (2000), pp. 7–13
6. R. Archana, A. Namasivayam. Effect of Ocimum sanctum on noise induced changes in neutrophil functions. J. Ethnopharmacol., 73 (2000), pp. 81–85
7. S.R. Vani, S.F. Cheng, C.H. Chuah. Comparative study of volatile compounds from genus Ocimum. Am. J. Appl. Sci., 6 (2009), pp. 523–528

 
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