Wild carrot pentane-based fractions suppress proliferation of human HaCaT keratinocytes and protect against chemically-induced skin cancer.

BACKGROUND: Previous studies in our laboratory showed that the Lebanese Daucus carota ssp. carota (wild carrot) oil extract possesses in vitro and in vivo anticancer activities. The present study aims to examine the cytotoxic effect of Daucus carota oil fractions on human epidermal keratinocytes and evaluate the chemopreventive activity of the pentane diethyl ether fraction on DMBA/TPA induced skin carcinogenesis in mice. METHODS: Wild carrot oil extract was chromatographed to yield four fractions (F1, 100% pentane; F2, 50:50 pentane:diethyl ether; F3, 100% diethyl ether; F4 93:7 chloroform:methanol). The cytotoxic effect of fractions (10, 25, 50 and 100 µg/mL) was tested on human epidermal keratinocytes (non-tumorigenic HaCaT cells and tumorigenic HaCaT-ras variants) using WST a ssay. Cell cycle phase distribution of tumorigenic HaCaT-ras variants was determined by flow cytometry post-treatment with F2 fraction. Apoptosis related proteins were also assessed using western blot. The antitumor activity of F2 fraction was also evaluated using a DMBA/TPA induced skin carcinoma in Balb/c mice. RESULTS: All fractions exhibited significant cytotoxicity, with HaCaT cells being 2.4-3 times less sensitive than HaCaT-ras A5 (benign tumorigenic), and HaCaT-ras II4 (malignant) cells. GC-MS analysis revealed the presence of a major compound (around 60%) in the pentane/diethylether fraction (F2), identified as 2-himachalen-6-ol. Treatment of HaCaT-ras A5 and HaCaT-ras II4 cells with F2 fraction resulted in the accumulation of cells in the sub-G1 apoptotic phase and decreased the population of cells in the S and G2/M phases. Additionally, F2 fraction treatment caused an up-regulation of the expression of pro-apoptotic (Bax) and down-regulation of the expression of anti-apoptotic (Bcl2) proteins. A decrease in the phosphorylation of AKT and ERK was also observed. Intraperitoneal treatment with F2 fraction (50 or 200 mg/kg) in the DMBA/TPA skin carcinogenesis mouse model showed a significant inhibition of papilloma incidence (mice with papilloma), yield (number of papilloma/mouse) and volume (tumor relative size) at weeks 15, 18 and 21. CONCLUSION: The present data reveal that F2 fraction has a remarkable antitumor activity against DMBA/TPA-induced skin carcinogenesis, an effect that may be mediated through inhibition of the MAPK/ERK and PI3K/AKT pathways.

Enhanced cellular uptake and photochemotherapeutic potential of a lipophilic strained Ru(ii) polypyridyl complex.

The use of ruthenium complexes as chemotherapeutic agents has been recently explored as one of the alternatives to conventional treatments. In the present study, two Ru(ii) polypyridyl complexes were synthesized and characterized: a strained [Ru(bipy)2(BC)]Cl2 (complex 1) where [bipy = 2,2'-bipyridine and BC = bathocuproine] along with the unstrained control [Ru(bipy)2(phen)]Cl2 (complex 2) where [phen = 1,10-phenanthroline]. The photophysical and photochemical analyses proved that unlike the photostable complex 2, complex 1 ejected both bipy and BC ligands at a ratio of 3 : 1 respectively. Results showed that the activity of complex 1 was significantly enhanced upon photoactivation. The response was however particularly significant in B16-F10 melanoma cells where phototoxicity index (PI = IC50 dark/IC50 light) was >900. When compared to cisplatin, the photoproducts were more potent against all tested cell lines, implying that the complex acquired significant chemotherapeutic potential upon irradiation. Cellular uptake of complex 1 and the free BC ligand were found to be significantly facilitated as evidenced by 400-600 fold increase in concentration of the compounds inside the cells relative to the extracellular culture medium. Complex 2 exhibited 35 times lower cellular concentration relative to complex 1. Flow cytometry and plasmid DNA gel electrophoresis measurements showed that complex 1 interacts with DNA inducing apoptosis in the dark and either late-apoptosis or necrosis upon irradiation. These findings corroborate the importance of lipophilic ligands such as BC to enhance uptake and subsequently improve the photochemotherapy potential of Ru(ii) polypyridyl complexes.

ß-2-Himachalen-6-ol inhibits 4T1 cells-induced metastatic triple negative breast carcinoma in murine model.

ß-2-himachalen-6-ol (HC), a major sesquiterpene isolated from the Lebanese wild carrot umbels, was shown to possess remarkable in vitro and in vivo anticancer activities. The present study investigates the anti-metastatic activity of HC post 4T1 breast cancer cells inoculation in a murine model. The effect of HC on 4T1 cell viability was assessed using WST-1 kit, while cell cycle analysis was performed using flow cytometry. Tumor development and metastasis were evaluated by injecting 4T1 cells in the mice mammary gland region followed by either HC or cisplatin treatment. The 6-thioguanine assay was used for the quantification of metastatic cells in the blood. HC treatment caused a dose-dependent decrease in cell viability with IC50 and IC90 values of 7 and 28 µg/mL respectively. Concomitant treatment with cisplatin significantly reduced cell viability when compared to cells treated with cisplatin or HC alone. Flow cytometry revealed a significant increase (p˂0.05) in cell count in the Sub-G1 phase at HC 10 µg/mL, and total DNA fragmentation (p˂0.001) at HC 25 µg/mL. Annexin/PI staining showed early and late apoptotic mode of cell death upon treatment with HC. Histopathological evaluation revealed less incidence of primary and metastatic tumor/inflammation in the HC and cisplatin treated groups. Tumor size and colony-forming units were significantly decreased in the HC treated group. HC treatment induced cell cycle arrest, promoted apoptosis and reduced the incidence of primary and metastatic lesions caused by 4T1 cells. The present findings suggest that HC has an anti-metastatic potential against aggressive types of cancer.

The chemotherapeutic effect of ß-2-himachalen-6-ol in chemically induced skin tumorigenesis.

ß-2-himachalen-6-ol (HC), a novel sesquiterpene derived from Lebanese wild carrot, was shown to possess a remarkable anticancer activity. The present study investigates the in vitro anticancer activity of HC and its effect on papillomas induced using a DMBA/TPA skin carcinogenesis mouse model. HaCaT-ras II-4 epidermal squamous cell viability was assessed using WST-1 kit. Cell cycle was analyzed by flow cytometry, and pro/anti-apoptotic proteins were measured using western blot. Mice papillomas were induced by DMBA and promoted with TPA for 18 weeks. At week 12, animals were divided into four groups: HC topically treated (5%Top), HC intraperitoneally treated (25 mg/kg; HC25), Cisplatin treated (2.5 mg/kg), and control (DMSO treated). Papilloma yield, volume, histology, and mice weight and liver function were assessed. HC treatment decreased significantly cell survival (IC50 = 7 and IC90 = 40 µg/ml) and increased significantly cells undergoing late apoptosis and necrosis. It also significantly decreased the levels of pro-caspase-3, p53, Bcl-2, p-Erk/Erk and p-Akt/Akt and increased p21 and Bax proteins. Treatment with HC25, HC5%Top or Cisplatin showed a significant decrease in papilloma yield and volume. Only Cisplatin treatment caused a significant decrease in body weight and increase in serum ALT. In conclusion, ß-2-himachalen-6-ol induced significant tumor shrinkage, an effect partly mediated via promoting apoptosis through inhibition of the MAPK/ERK and PI3K/AKT pathways, with no significant toxicity to laboratory mice.

ß-2-himachalen-6-ol protects against skin cancer development in vitro and in vivo.

BACKGROUND: Previous studies in our laboratory showed that Daucus carota oil extract (DCOE) possesses remarkable in-vitro anticancer activity and antitumour promoting effect against DMBA/TPA skin carcinogenesis in mice. Chemical analysis of DCOE led to the isolation of the ß-2-himachalen-6-ol (HC), major sesquiterpene with a potent anticancer activity against various colon, breast, brain and skin cancer cells. This study investigated the anticancer activity of HC against invasive epidermal squamous cell carcinoma cells and evaluated its effect in a DMBA/TPA skin carcinogenesis Balb/c murine model. METHODS: HaCaT-ras II-4 epidermal squamous cells were treated with HC (1, 5, 10, 25 and 50 µg/ml), and cell viability was evaluated with WST 1 assay kit. Cell cycle analysis was carried out by flow cytometry, and pro/anti-apoptotic proteins were measured using Western blot. The effect of topical and intraperitoneal (IP) treatment with HC in mice was assessed using the DMBA/TPA skin carcinogenesis model. Cisplatin (2.5 mg/kg; IP) was used as a positive control. Papilloma incidence, yield and volume were monitored, and isolated papillomas were assessed for their pro/anti-apoptotic proteins and morphology. RESULTS: ß-2-himachalen-6-ol showed a dose-dependent decrease in cell survival with an IC50 and IC90 of 8 and 30 µg/ml, respectively. Flow cytometry analysis revealed that treatment with 10 µg/ml HC significantly increased the number of cells undergoing late apoptosis (28%), while 25 µg/ml caused a larger cell shift towards late apoptosis (46.6%) and necrosis (39%). A significant decrease in protein levels of p53 and Bcl-2 and a significant increase in p21 and Bax were observed. Also, there was a significant decrease in p-Erk and p-Akt protein levels. The treatment of mice (IP and topical) with HC caused a significant decrease in papilloma yield, incidence and volume. Similar effects were observed with cisplatin treatment, but HC-treated groups exhibited twofold to threefold increase in survival rates. Similar patterns in the pro- and anti-apoptotic proteins were observed in mice treated with HC, except for a significant increase in p53 protein. CONCLUSIONS: In conclusion, HC treatment induced cell cycle arrest (low dose) and promoted apoptosis partly via inhibition of the MAPK/ERK and PI3K/AKT pathways with no significant toxicity to laboratory mice.

Antitumor activity of ß-2-himachalen-6-ol in colon cancer is mediated through its inhibition of the PI3K and MAPK pathways.

Previous studies in our laboratory showed that Daucus carota oil extract (DCOE) possesses in vitro and in vivo anticancer activities. Chemical analysis of DCOE led to the isolation of ß-2-himachalen-6-ol (HC) which exhibited potent anticancer activity against colon, breast, brain and skin cancer cells. The present study investigates the anticancer activity of HC against SW1116 colon cancer cell lines, and evaluates its effect in a 1,2-dimethylhydrazine (DMH) colon carcinogenesis black6 mice model. The SW1116 colon cancer cell line was treated with HC (1, 5, 10 and 25 µg/ml) and cell viability was evaluated with WST 1 assay kit. Cell cycle analysis was carried out by flow cytometry, and pro/anti-apoptotic proteins were measured using western blot. The effect of intraperitoneal (IP) treatment with HC (10, 25 and 50 µg/ml) in mice was assessed using the DMH colon carcinogenesis model with Cisplatin (2.5 µg/kg; IP) as a positive control. Blood samples were collected for assessment of liver toxicity and colon tumor incidence and size were studied histologically. HC showed a dose-dependent decrease in cell survival with an IC50 of 18 and 14.5 µg/ml after 24 and 48 h respectively. Flow cytometry analysis revealed that 10 µg/ml HC increased the number of cells undergoing necrosis (18.05%) and late apoptosis (15.66%). At HC 25 µg/ml more cells shifted toward necrosis (58.01%) and late apoptosis (30.47%). Western blot analysis revealed a significant decrease in p-Erk, p-Akt, pro-caspase-3 and Bcl-2 and an increase in p53, p21, Bax and PARP proteins. Mice treatment (IP) with HC caused a significant decrease in tumor incidence and size. Similar effects were observed with cisplatin treatment. In conclusion, HC treatment (low dose) induced cell cycle arrest and promoted apoptosis via inhibition of the MAPK/ERK and PI3K/AKT pathways. HC treatment also had antitumor effect in vivo with no significant toxicity to laboratory mice.