Citral induced apoptosis in MDA-MB-231 spheroid cells

Background: Breast cancer remains a leading cause of death in women worldwide. Although breast cancer therapies have greatly advanced in recent years, many patients still develop tumour recurrence and metastasis, and eventually succumb to the disease due to chemoresistance. Citral has been reported to show cytotoxic effect on various cancer cell lines. However, the potential of citral to specifically target the drug resistant breast cancer cells has not yet been tested, which was the focus of our current study. Methods: The cytotoxic activity of citral was first tested on MDA-MB-231 cells in vitro by MTT assay. Subsequently, spheroids of MDA-MB-231 breast cancer cells were developed and treated with citral at different concentrations. Doxorubicin, cisplatin and tamoxifen were used as positive controls to evaluate the drug resistance phenotype of MDA-MB-231 spheroids. In addition, apoptosis study was performed using AnnexinV/7AAD flowcytometry. Aldefluor assay was also carried out to examine whether citral could inhibit the ALDH-positive population, while the potential mechanism of the effect of citral was carried out by using quantitative real time- PCR followed by western blotting analysis. Results: Citral was able to inhibit the growth of the MDA-MB-231 spheroids when compared to a monolayer culture of MDA-MB-231 cells at a lower IC50 value. To confirm the inhibition of spheroid self-renewal capacity, the primary spheroids were then cultured to additional passages in the absence of citral. A significant reduction in the number of secondary spheroids were formed, suggesting the reduction of self-renewal capacity of these aldehyde dehydrogenase positive (ALDH+) drug resistant spheroids. Moreover, the AnnexinV/7AAD results demonstrated that citral induced both early and late apoptotic changes in a dose-dependent manner compared to the vehicle control. Furthermore, citral treated spheroids showed lower cell renewal capacity compared to the vehicle control spheroids in the mammosphere formation assay. Gene expression studies using quantitative real time PCR and Western blotting assays showed that citral was able to suppress the self-renewal capacity of spheroids and downregulate the Wnt/β-catenin pathway. Conclusion: The results suggest that citral could be a potential new agent which can eliminate drug-resistant breast cancer cells in a spheroid model via inducing apoptosis

Cytotoxic Effects of Ethanol Extract of Malaysia Indigenous Marine Microalga, Chaetoceros Calcitrans on Human Breast Canser Line MCF-7

Algae are being used by millions of humans and animals around the world as nutritional or pharmaceutical ingredients due to their vast diversity, diverse chemical compositions and biological activities. In the current study, cytotoxic effects of Malaysian a marine microalga, Chaetoceros calcitrans were evaluated. Briefly, Chaetoceros calcitrans was identified and cultured. The Chaetoceros calcitrans ethanol extract (EEC) was screened using MTT assay for cytotoxicity on various human cancer cell lines, K562 (leukemia), HT29 (colorectal), HeLa (cervical), MCF-7 (breast) and also on normal cells, MCF10A (human normal breast cell) and PBMC (peripheral blood monolayer cell). Tamoxifen was used as a positive control. The mode of cell death induced by EEC was determined by Annexin V/PI followed by flow cytometry cell cycle analysis. The effects of EEC treatment on MCF-7 cells in expressions of apoptotic related genes, Bax, Bcl-2, BCL2L1, TNF, P53, Fas, Caspase3, Caspase7 and Caspase9 and cell cycle genes, P21Cip1, CyclinA2, CDK2 and MDM2 were determined using GeXP system, a multiplex quantitative gene expression technology. Overall, the EEC showed different IC50 values on the tested cell lines at different time points (24, 48 and 72 hours). The lowest IC50 value for MCF-7 (3.00±0.65 μg/ml) compared to that for MCF-10A cells (12.00±0.59 μg/ml) was obtained at 24 hour after treatment. Besides that the IC50 value of EEC on MCF-7 (3.00±0.65 μg/ml) was lower than the IC50 value of Tamoxifen (12.00±0.52). EEC was not cytotoxic towards PBMC even at its highest concentration. Hence, the EEC can be considered having anti cancer properties. Based on Annexin V/PI and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells is through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increase compared to treated MCF-10A cells which showed only 6 and 7 folds increase at the same time points, respectively. The results from this study also showed that EEC induced apoptosis of MCF-7 cells without cell cycle arrest that is associated with the modulation of expression of selected cell cycle and apoptosis related genes such as MDM2, p21Cip1 as well as increasing the expression of pro-apoptotic protein Bax and decreasing the expression of the antiapoptotic protein Bcl-2. The EEC treatment on MCF-7 cells caused the fold changes of MDM2 to decrease from 1.8 at 6 hours to 1.4 at 24 hours; the fold changes of CyclinA2 also decreased from 3.5 at 6 hours to 1.5 at 24 hour whilst,the fold changes of p21Cip1 also increased from 0.8 at 6 hours to 1.9 at 24 hour. The EEC treated cells also modulate the fold changes of the key caspase molecules where the fold changes of caspase3 and caspase7 decreased from 1.5 and 2.1 at 6 hour to 1.3 and 2.0 at 24 hours, respectively. The Bax activation might have been involved in the release of cytochrome c from the mitochondria and clustered with APAF-1, a apoptotic protease activating factor 1 and resulted in activation of caspase9 (although it was not determined in the experiment) which then cleaved the downstream caspease3, 6 and 7 that lead to apoptosis. Hence, EEC induced apoptosis of MCF-7 cells associated with expression of several major regulators of the cell cycle such as CDK2, MDM2 and Cyclin A2 and apoptotic related genes by increasing in Bax/Bcl-2 ratio that in turn activate the caspase-dependent pathway. This study also showed that EEC could induce apoptosis through a caspase-dependent pathway by activating caspase3 and 7 in MCF-7 cells. Based on the results obtained from this study, it appears likely that EEC has the potential cytotoxic effect on breast cancer. However, more studies were required which include isolation, characterization and synthesis of the active compound (s) found in EEC that was associated with the strong apoptotic effects against breast cancer cells

In vivo anti-tumor effects of citral on 4T1 breast cancer cells via induction of apoptosis and downregulation of aldehyde dehydrogenase activity

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among females globally. The tumorigenic activities of cancer cells such as aldehyde dehydrogenase (ALDH) activity and differentiation have contributed to relapse and eventual mortality in breast cancer. Thus, current drug discovery research is focused on targeting breast cancer cells with ALDH activity and their capacity to form secondary tumors. Citral (3,7-dimethyl-2,6-octadienal), from lemon grass (Cymbopogon citrates), has been previously reported to have a cytotoxic effect on breast cancer cells. Hence, this study was conducted to evaluate the in vivo effect of citral in targeting ALDH activity of breast cancer cells. BALB/c mice were challenged with 4T1 breast cancer cells followed by daily oral feeding of 50 mg/kg citral or distilled water for two weeks. The population of ALDH+ tumor cells and their capacity to form secondary tumors in both untreated and citral treated 4T1 challenged mice were assessed by Aldefluor assay and tumor growth upon cell reimplantation in normal mice, respectively. Citral treatment reduced the size and number of cells with ALDH+ activity of the tumors in 4T1-challenged BALB/c mice. Moreover, citral-treated mice were also observed with smaller tumor size and delayed tumorigenicity after reimplantation of the primary tumor cells into normal mice. These findings support the antitumor effect of citral in targeting ALDH+ cells and tumor recurrence in breast cancer cells

Cytotoxic effect of ethanol extract of microalga, Chaetoceros calcitrans, and its mechanisms in inducing apoptosis in human breast cancer cell line

Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00±0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00±0.52 g/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug

Arisaema atrolinguum F. Maek.

原著和名: クロハシテンナンシャウ科名: サトイモ科 = Araceae採集地: 静岡県 賀茂郡 河津町 七滝 (伊豆 河津町 七滝)採集日: 1972/4/5採集者: 萩庭丈壽整理番号: JH006040国立科学博物館整理番号: TNS-VS-95604

Effect of compound C1 on the cell cycle distribution of MCF-7 cells.

<p>(<b>A</b>) Shows the untreated MCF-7 cells (control). After treatment with the IC₅₀ concentration of complex C1 for (<b>B</b>) 24, (<b>C</b>) 48 and (<b>D</b>) 72 hours, a flow cytometric analysis was performed on treated and untreated MCF-7 cells (<b>E</b>). <b>(F)</b> Shows the protein expression of the G0/G1 phase specific cyclins p21 and p27. The quantitative analysis indicated cell cycle arrest at the G0/G1 phase. Data are expressed as the mean ± the standard deviation. Each independent experiment included triplicate treatment groups. Statistical significance was equal to *<i>p</i> < 0.05.</p

Citral induced apoptosis in MDA-MB-231 spheroid cells

Abstract Background Breast cancer remains a leading cause of death in women worldwide. Although breast cancer therapies have greatly advanced in recent years, many patients still develop tumour recurrence and metastasis, and eventually succumb to the disease due to chemoresistance. Citral has been reported to show cytotoxic effect on various cancer cell lines. However, the potential of citral to specifically target the drug resistant breast cancer cells has not yet been tested, which was the focus of our current study. Methods The cytotoxic activity of citral was first tested on MDA-MB-231 cells in vitro by MTT assay. Subsequently, spheroids of MDA-MB-231 breast cancer cells were developed and treated with citral at different concentrations. Doxorubicin, cisplatin and tamoxifen were used as positive controls to evaluate the drug resistance phenotype of MDA-MB-231 spheroids. In addition, apoptosis study was performed using AnnexinV/7AAD flowcytometry. Aldefluor assay was also carried out to examine whether citral could inhibit the ALDH-positive population, while the potential mechanism of the effect of citral was carried out by using quantitative real time- PCR followed by western blotting analysis. Results Citral was able to inhibit the growth of the MDA-MB-231 spheroids when compared to a monolayer culture of MDA-MB-231 cells at a lower IC50 value. To confirm the inhibition of spheroid self-renewal capacity, the primary spheroids were then cultured to additional passages in the absence of citral. A significant reduction in the number of secondary spheroids were formed, suggesting the reduction of self-renewal capacity of these aldehyde dehydrogenase positive (ALDH+) drug resistant spheroids. Moreover, the AnnexinV/7AAD results demonstrated that citral induced both early and late apoptotic changes in a dose-dependent manner compared to the vehicle control. Furthermore, citral treated spheroids showed lower cell renewal capacity compared to the vehicle control spheroids in the mammosphere formation assay. Gene expression studies using quantitative real time PCR and Western blotting assays showed that citral was able to suppress the self-renewal capacity of spheroids and downregulate the Wnt/β-catenin pathway. Conclusion The results suggest that citral could be a potential new agent which can eliminate drug-resistant breast cancer cells in a spheroid model via inducing apoptosis