Kotamballi N. Chidambara Murthy

The natural alkaloid berberine targets multiple pathways to induce cell death in cultured human colon cancer cells.

In the current paper, berberine, an isoquinoline alkaloid, was tested for its chemopreventive potential in colon cancer (SW480) cells. Berberine inhibited proliferation of colon cancer cells in a dose- and time-dependent manner. Interestingly, this compound exhibited minimum toxicity in normal cells at 200 μM. Berberine arrested SW480 cell cycle at G2/M phase, which was supported by induction of p21 expression. Induction of a series of biochemical events, including loss of mitochondrial membrane potential, release of cytochrome-c to cytosol, induction of Bcl-2 family proteins, caspases and cleavage of poly (ADP-ribose) polymerase (PARP), by berberine suggests its ability to induce apoptosis. In addition, berberine also inhibited inflammation, as evidenced by induction of expression of NFκB and Cox(2). Furthermore, berberine inhibited caspase-8 mediated angiogenesis, as confirmed through expression of tumor necrotic factor related apoptosis-inducing ligand (TRAIL), vascular endothelial growth factor (VEGF) and survivin. The results of the current study demonstrated that berberine has the ability to cause cell cycle arrest, induce apoptosis and inhibit inflammation in colon cancer cells. The magnitude of the effects observed suggests that berberine may be worth considering for further studies of its potential applications for improving health, either as a preventative or a potential treatment.

Citrus Limonin and Its Glucoside Inhibit Colon Adenocarcinoma Cell Proliferation through Apoptosis

The current study was an attempt to elucidate the mechanism of human colon cancer cell proliferation inhibition by limonin and limonin glucoside (LG) isolated from seeds of Citrus reticulata. The structures of purified compounds were confirmed by NMR and quantified using HPLC. These compounds of more than 95% purity were subjected to proliferation inhibition assay using human colon adenocarcinoma (SW480) cells. The IC50 value of 54.74 and 37.39 μM was observed for limonin and LG, respectively at 72 h. Following confirmation of proliferation inhibition, pattern of DNA fragmentation and activation of caspase-3 of the cells treated with limonoids suggest involvement of apoptosis. Furthermore, reduction in the transcription ratio of bcl2/bax and induction of cytochrome c release from mitochondria to cytosol with treatment of limonoids confirm the activation of intrinsic apoptosis pathway. The activity of Bax and Bcl2 was confirmed through analysis of mitochondrial membrane potential and intracellular calcium in the cells treated with limonin and LG; the net content of caspase-8 was not affected by limonoids. Results of the current study provide compelling evidence on the induction of mitochondria mediated intrinsic apoptosis by both limonin and LG in cultured SW480 cells for the first time.

D-limonene rich volatile oil from blood oranges inhibits angiogenesis, metastasis and cell death in human colon cancer cells

AIMS To identify the chemical constituents of volatile oil from blood orange (Citrus sinensis (L) Osbeck) and understand the possible mechanisms of inhibition of colon cancer cell proliferation. MAIN METHODS Volatile oil was obtained from blood oranges by hydro-distillation. Nineteen compounds were identified by GC-MS and d-limonene was found to be the major component. The blood orange volatile oil was formulated into an emulsion (BVOE) and examined for its effects on viability of colon cancer cells. In addition, experiments were performed to understand the possible mechanism of proliferation inhibition, angiogenesis and metasasis by BVOE. KEY FINDINGS BVOE exhibited dose-dependent inhibition of cell proliferation and induced apoptosis in the colon cancer cells, as confirmed by flow cytometry. Immunoblotting of colon cancer cells treated with BVOE shows dose-dependent induction of Bax/Bcl2) and inhibition of vascular endothelial growth factor (VEGF). Furthermore, treatment of serum starved SW480 and HT-29 cells with 100μg/ml BVOE suggested the inhibition of VEGF and markers associated with inhibition of angiogenesis. The antiangiogenic activity of BVOE was also confirmed by inhibition of in vitro tube formation in human umbilical vein endothelial cells. Dose-dependent anti-metastasis activity and blockage of vascular endothelial growth factor receptor 1 (VEGFR1) binding following treatment with BVOE were confirmed by cell migration assays and immunoblots to detect decreased expression of matrix metalloproteinases (MMP-9). SIGNIFICANCE The results of this study provide persuasive evidence of the apoptotic and anti-angiogenesis potential of BVOE in colon cancer cells. The extent of induction of apoptosis and inhibition of angiogenesis suggest that BVOE may offer great potential for prevention of cancer and may be appropriate for further studies.

Citrus limonoids and curcumin additively inhibit human colon cancer cells

In the current study, we examined the ability of limonoids, including limonin, limonin glucoside (LG) and curcumin, to inhibit proliferation of human colon cancer (SW480) cells. Additionally, we studied the effect of combining these two classes of natural compounds on inhibition of proliferation and the possible mode of cytotoxicity. The SW480 cells were treated with compounds individually and in combination to understand the effect on cell death, DNA fragmentation, caspase-3 activity and the expression of Bax, Bcl-2 and caspase-3 proteins. Results of cell proliferation assays suggest that combinations of limonoids with curcumin at three different ratios (1 : 3, 1 : 1 and 3 : 1) to a final concentration of 50 ppm demonstrated up to 96% inhibition of cell proliferation. The MTT assay results were also confirmed by counting viable cells. Further, incubation of cells with combinations of limonoids and curcumin resulted in elevation of total cellular caspase-3 activity by 3.5-4.0 fold along with a 2- to 4-fold increase in the Bax/Bcl-2 ratio. The expression of pro-caspase-3 and its cleaved products in cells treated with curcumin (individually or combination) indicates higher potency of the combination to induce apoptosis. For the first time, this study provides compelling evidence of the pharmacodynamic additive effect of limonoids and curcumin in inhibiting human colon cancer cells. The above results were also confirmed by fluorescence microscopy of SW480 cells treated with limonoids, curcumin and combination, after tagging with fluorescent probes. These results suggest that consumption of curcumin and limonoids together may offer greater protection against colon cancer.

Stability of Betalain Pigments of Red Beet

Although pigments of biological origin are intensely researched for their market potentials and health benefits, their stability, from the maturity status of the source material to their processing and products into which the pigments are incorporated and the storage of those products, is shrouded with many problems. Both extrinsic and intrinsic factors affect the prospective arrays of applications of natural pigments such as betalains. Although the maturity of the red beet may not be the major quality trait in sourcing, the various processing steps, starting from the selection of the extraction medium, extraction conditions, product concentration, storage, transportation and incorporation into products, are huge tasks. The extrinsic factors affecting betalain stability include oxygen, temperature, pH, light and chemicals of processing and food products. On the other hand, the intrinsic factors in the red beet, apart from the quality of the source material, are mainly enzymes: β-glucosidases, polyphenoloxidases (PPOs) and peroxidases (PODs). The current chapter is a compilation of research progress made in understanding the factors affecting the stability of betalains, the chemical changes taking place during degradation and their further fate. Improvements in processing/extraction techniques and storage conditions that impart stability to betalain pigments are discussed. Additionally, application of fermentation and other advanced processes used for the preparation of pigments and food formulations are discussed.

Apoptosis mediated cytotoxicity of citrus obacunone in human pancreatic cancer cells.

Obacunone is one of the oxygenated triterpenoids found in rutaceae family. It has been demonstrated for various biological activities including inhibition of cancer cells proliferation and tumor. The current study is an attempt to understand the mode of cytotoxicity of obacunone using cultured pancreatic cancer (MDA Panc-28) cells. Obacunone exhibited dose and time dependant inhibition of Panc-28 cells proliferation . This was also associated with phosphatidylserine translocation from inner surface of plasma membrane to outer surface in the treated cells, suggesting the involvement of apoptosis. Analysis of cells treated with 50 and 100 μM of obacunone suggests activation of initiator caspase-9 and effector caspase-3, indicating involvement of caspases induced apoptosis. Obacunone upregulated expression of tumor suppressor protein p53, pro-apoptotic protein Bax and downregulated anti-apoptotic protein Bcl2. Furthermore, release of cytochrome-c into cytosol was observed in the cells treated with obacunone. It also resulted in down regulation of vital inflammatory mediators such as NFκB and Cox-2 suggesting the anti-inflammatory potential. For the first time, current study provides an evidence on activation of caspase dependant, cytochrome-c mediated intrinsic apoptosis and anti-inflammatory activity in Panc-28 cells by citrus obacunone. Induction of cytotoxicity and apoptosis was also confirmed by fluorescent tagged microscopic images of obacunone treated Panc-28 cells.

Inhibition of prostate cancer (LNCaP) cell proliferation by volatile components from Nagami kumquats.

Fresh Nagami kumquats (Fortunella margarita) were subjected to hydrodistillation using a Clevenger-type apparatus to obtain volatile oil. The chemical composition of the volatile oil was analyzed by GC-MS using Rtx-5 Sil MS and DB Wax columns. A total of 25 volatile compounds were identified by mass spectra, retention index, and comparison with known standards. The major identified compounds are d-limonene (41.64 %), β-myrecene (16.54 %), linalyl propionate (9.55 %), and germacrene-D (5.93 %) from the Rtx-5 Sil MS column; d-limonene and β-myrecene were also separated as major compounds on the DB wax column. The oil is rich in hydrocarbons (77.41 %) consisting of 60.05 % monoterpenes and 17.36 % sesquiterpenes. Interestingly, oxygenated hydrocarbons (17.6 %) were also found in kumquat volatile oil. Certain volatile compounds were also confirmed by positive chemical ionization and NMR spectra. Further, the volatile oil demonstrated good DPPH radical scavenging activity and antioxidant capacity. Kumquat volatile oil at 200 ppm concentration exhibited 55 %, 61 %, and 63.4 % inhibition of human prostate cancer (LNCaP) cell proliferation at 24, 48, and 72 h, respectively, by cell count assays. Significant increases in expression of bax/bcl2 and p53 proteins confirmed that volatile oil induces apoptosis. In addition, inhibition of inflammatory markers such as NF-κB and Cox-2 was observed. The cleavage of caspase-8 in the LNCaP cells treated with volatile oil demonstrated that apoptosis occurred through an extrinsic pathway. This is the first report of the identification and possible mechanisms of in vitro antiproliferative effects of kumquat volatile components on human prostate cancer (LNCaP) cells.

5-Geranyloxy-7-methoxycoumarin inhibits colon cancer (SW480) cells growth by inducing apoptosis.

For the first time, three coumarins were isolated from the hexane extract of limes (Citrus aurantifolia) and purified by flash chromatography. The structures were identified by NMR (1D, 2D) and mass spectral analyses as 5-geranyloxy-7-methoxycoumarin, limettin, and isopimpinellin. These compounds inhibited human colon cancer (SW-480) cell proliferation, with 5-geranyloxy-7-methoxycoumarin showing the highest inhibition activity (67 %) at 25 µM. Suppression of SW480 cell proliferation by 5-geranyloxy-7-methoxycoumarin was associated with induction of apoptosis, as evidenced by annexin V staining and DNA fragmentation. In addition, 5-geranyloxy-7-methoxycoumarin arrested cells at the G0/G1 phase, and induction of apoptosis was demonstrated through the activation of tumour suppressor gene p53, caspase8/3, regulation of Bcl2, and inhibition of p38 MAPK phosphorylation. These findings suggest that 5-geranyloxy-7-methoxycoumarin has potential as a cancer preventive agent.

Cytotoxicity of obacunone and obacunone glucoside in human prostate cancer cells involves Akt-mediated programmed cell death

Obacunone and obacunone glucoside (OG) are naturally occurring triterpenoids commonly found in citrus and other plants of the Rutaceae family. The current study reports the mechanism of cytotoxicity of citrus-derived obacunone and OG on human androgen-dependent prostate cancer LNCaP cells. Both limonoids exhibited time- and dose-dependent inhibition of cell proliferation, with more than 60% inhibition of cell viability at 100 μM, after 24 and 48 h. Analysis of fragmentation of DNA, activity of caspase-3, and cytosolic cytochrome-c in the cells treated with limonoids provided evidence for activation of programmed cell death by limonoids. Treatment of LNCaP cells with obacunone and OG resulted in dose-dependent changes in expression of proteins responsible for the induction of programmed cell death through the intrinsic pathway and down-regulation of Akt, a key molecule in cell signaling pathways. In addition, obacunone and OG also negatively regulated an inflammation-associated transcription factor, androgen receptor, and prostate-specific antigen, and activated proteins related to the cell cycle, confirming the ability of limonoids to induce cytotoxicity through multiple pathways. The results of this study provided, for the first time, an evidence of the cytotoxicity of obacunone and OG in androgen-dependent human prostate cancer cells.

Enhanced colon cancer chemoprevention of curcumin by nanoencapsulation with whey protein.

To improve bioavailability and enhance colon cancer prevention ability of curcumin, whey protein was used to nanoencapsulate at three different ratios such as 70:30, 50:50 and 35:65 for the first time. The drug loading, entrapment efficiency and structural changes of curcumin was confirmed by quantitative NMR spectroscopy. The nanoparticles prepared using the three ratios had an average diameters of 236.5±8.8, 212±3.4, and 187±11.4nm, as well as zeta (ζ) potentials of -13.1,-9.26, and -4.63mV, respectively, at pH 7.0. The cytotoxicity assay was performed for human colon and prostate cancer (SW480 and LNCap) by MTT assay and results showed significantly higher cytotoxicity of nanoencapsulated curcumin (NEC) (equivalent to 30.91, 20.70 and 16.86µM of NEC-1, 2 and 3 respectively), as compared to plain curcumin at 50µM after 72h of treatment. Cytotoxicity was also confirmed by microscopy of treated cells stained with acridine orange and propidium iodide. The cells treated with 50µM of curcumin, 30.91µM (NEC-1), 20.70µM (NEC-2) and 16.86µM (NEC-3) showed enhanced activation of p53 and elevated bax/Bcl2 expression (NEC-3), increased cytochrome-c in cytosol (NEC-2) confirming the enhanced cytotoxicity. To confirm the increased bioavailability, the intracellular curcumin was measured using fluorescence intensity. The fluorescent signal for intracellular curcumin was increased by 12, 30, and 21% for NEC-1, NEC-2, and NEC-3 respectively as compared to plain curcumin at 4h. Based on these results, we conclude that nanoencapsulated curcumin with whey protein will have potential to be considered for clinical applications for future studies.