Cinnamon extract induces tumor cell death through inhibition of NFkappaB and AP1

Abstract

Background: Cinnamomum cassia bark is the outer skin of an evergreen tall tree belonging to the family Lauraceae containing several active components such as essential oils (cinnamic aldehyde and cinnamyl aldehyde), tannin, mucus and carbohydrate. They have various biological functions including anti-oxidant, anti-microbial, anti-inflammation, anti-diabetic and anti-tumor activity. Previously, we have reported that anti-cancer effect of cinnamon extracts is associated with modulation of angiogenesis and effector function of CD8+ T cells. In this study, we further identified that anti-tumor effect of cinnamon extracts is also link with enhanced pro-apoptotic activity by inhibiting the activities NFkappaB and AP1 in mouse melanoma model.

Methods: Water soluble cinnamon extract was obtained and quality of cinnamon extract was evaluated by HPLC (High Performance Liquid Chromatography) analysis. In this study, we tested anti-tumor activity and elucidated action mechanism of cinnamon extract using various types of tumor cell lines including lymphoma, melanoma, cervix cancer and colorectal cancer in vitro and in vivo mouse melanoma model.

Results: Cinnamon extract strongly inhibited tumor cell proliferation in vitro and induced active cell death of tumor cells by up-regulating pro-apoptotic molecules while inhibiting NFkappaB and AP1 activity and their target genes such as Bcl-2, BcL-xL and survivin. Oral administration of cinnamon extract in melanoma transplantation model significantly inhibited tumor growth with the same mechanism of action observed in vitro.

Conclusion: Our study suggests that anti-tumor effect of cinnamon extracts is directly linked with enhanced pro-apoptotic activity and inhibition of NFkappaB and AP1 activities and their target genes in vitro and in vivo mouse melanoma model. Hence, further elucidation of active components of cinnamon extract could lead to development of potent anti-tumor agent or complementary and alternative medicine for the treatment of diverse cancers.

Figure 1

Treatment of cinnamon extract inhibits the growth of various cancer cells. Cinnamon (0.5 mg/ml) was treated for 0, 48 and 72 hrs to cancer cell lines (Hela; cervical cancer, Caco2; colon cancer, Clone M3; melanoma and B16F10; melanoma). After treatment of cinnamon extract, (A) morphological changes of each cancer cell lines were monitored by microscopic observation. (B) Proliferation and viability of cancer cells were measured by cell viability assay at the indicated time points. Error bars indicated SD. Data are representative of three independent experiments.

Figure 2

Cinnamon extract treatment induces active cell death in melanoma cells. At the indicated time periods after treatment of cinnamon extract, cells were stained with Annexin V and 7-ADD. Double positive (Annexin V⁺ and 7-ADD⁺) (A) or Annexin V⁺ positive (B) populations were analyzed by FACS. Gene expression (C) and protein (D) level of pro-apoptotic molecules such as Bad, Bim, Bax and Bak were measured by quantitative real-time PCR or immunoblotting, respectively. Error bars indicated SD. One (*), two (**) or three asterisks (***) indicate p < 0.05, p < 0.005 or p < 0.001, respectively. Data are representative of three independent experiments.

Figure 3

Cinnamon extract treatment down-regulates the levels of NFκB and AP1 and their target genes. (A) After treatment of cinnamon extract for indicated time periods, the levels of NFκB and AP1 in total cell lysates (left) and nucleus extracts (right) were compared by immunoblotting. The effect of cinnamon extract on the activity NFκB (B) and AP1 (C) was measured by reporter assay. B16F10 cells were transfected with AP1- or NFκB-dependent reporter construct and then stimulated with PMA and ionomycin (P+I) for 4 hours in the absence or presence of cinnamon extract (CE). Gene expression (D) and protein (E) levels of anti-apoptotic genes were measured by quantitative real-time PCR or immunoblotting, respectively. Data are representative of three independent experiments with similar results. One (*), two (**) or three asterisks (***) indicate p < 0.05, p < 0.005 or p < 0.001, respectively.

Figure 4

Oral administration of cinnamon extract inhibits melanoma growth in vivo. Ten days after melanoma transplantation, cinnamon extract (CE; 400 μg/g mouse weight) or PBS (Cont) was orally administrated every two days for 20 days. (A) Photographs of representative tumors from each group at day 14 and 30. During the treatment period, the tumor volumes (B) and tumors weights (C) and survivor rate (D) in each group were daily measured. Data are representative of three independent experiments (10 mice/each group). One (*) or two (**) asterisks indicates p < 0.05 or p < 0.005, respectively. Data are representative of three individual experiments.

Figure 5

Cinnamon extract treatment induces tumor apoptosis by decreasing the levels of NFκB and AP1 and their target genes. (A) Genomic DNA was isolated from tumor tissues from each treatment group and DNA fragmentation was confirmed by staining with ethidium bromide in 2% agarose gel. (B) The protein levels of pro-caspase and active caspases 3 in tumor tissues from each group were determined by immunoblotting. (C) To check the structural changes of nucleus, tumor tissues from each group were sectioned and then stained with Hoechst. Round areas and arrows indicate apoptotic cells (bigger size of this picture was included in Additional file 4, Figure S3). (D) The levels of NFκB and AP1 in tumor tissues from each group were compared by immunoblotting between the treatment groups. The gene expression (E) and protein (F) level of Bcl-2, BcL-xL and survivin in tumor tissues were measured by quantitative real-time PCR or immunoblotting, respectively. Two (**) asterisk indicates p < 0.005. Not significant (NS). Data are representative of three individual experiments with similar results.