The Yemeni Brown Algae Dictyota dichotoma Exhibit High In Vitro Anticancer Activity Independent of Its Antioxidant Capability.

The aim of this study was to investigate the anticancer and antioxidant activities as well as the safety of the brown algae Dictyota dichotoma of the Western seacoast of Yemen. Cytotoxicity of methanol extract of D. dichotoma and several of its fractions, petroleum ether, chloroform, ethyl acetate, n-butanol, and aqueous extracts against seven different cancer cell lines was determined by crystal violet staining. The antioxidant activity was also assessed using the DPPH radical scavenging assay. Acute toxicity study was performed on rats at increasing doses of the methanol extract. Extracts of D. dichotoma exerted a significant dose-dependent cytotoxicity on the seven tumor cell lines but were generally more selective on MCF-7 and PC-3. Among all fractions, the chloroform fraction of the D. dichotoma displayed the highest cytotoxic activity and was most effective in MCF-7, PC3, and CACO cells (IC50 = 1.93 ± 0.25, 2.2 ± 0.18, and 2.71 ± 0.53 µg/mL, respectively). The petroleum ether fraction was also effective, particularly against MCF-7 and PC-3 (IC50 = 4.77 ± 0.51 and 3.93 ± 0.51 µg/mL, respectively) whereas the activity of the ethyl acetate fraction was more pronounced against HepG2 and CACO (IC50 = 5.06 ± 0.21 and 5.06 ± 0.23 µg/mL, respectively). Of all the extracts tested, the crude methanolic extract of the algae exhibited only a modest antioxidant potential (IC50 = 204.6 ± 8.3 µg/mL). Doses as high as 5000 mg/kg body weight of D. dichotoma methanolic extracts were safe and well tolerated by rats. The overall results showed that D. dichotoma exhibited a significant cytotoxic activity probably due to the occurrence of nonpolar cytotoxic compounds, which is independent of its antioxidant capability.

Transcription factor-induced activation of cardiac gene expression in human c-kit+ cardiac progenitor cells.

Although transplantation of c-kit+ cardiac progenitor cells (CPCs) significantly alleviates post-myocardial infarction left ventricular dysfunction, generation of cardiomyocytes by exogenous CPCs in the recipient heart has often been limited. Inducing robust differentiation would be necessary for improving the efficacy of the regenerative cardiac cell therapy. We assessed the hypothesis that differentiation of human c-kit+ CPCs can be enhanced by priming them with cardiac transcription factors (TFs). We introduced five different TFs (Gata4, MEF2C, NKX2.5, TBX5, and BAF60C) into CPCs, either alone or in combination, and then examined the expression of marker genes associated with the major cardiac cell types using quantitative RT-PCR. When introduced individually, Gata4 and TBX5 induced a subset of myocyte markers. Moreover, Gata4 alone significantly induced smooth muscle cell and fibroblast markers. Interestingly, these gene expression changes brought by Gata4 were also accompanied by morphological changes. In contrast, MEF2C and NKX2.5 were largely ineffective in initiating cardiac gene expression in CPCs. Surprisingly, introduction of multiple TFs in different combinations mostly failed to act synergistically. Likewise, addition of BAF60C to Gata4 and/or TBX5 did not further potentiate their effects on cardiac gene expression. Based on our results, it appears that GATA4 is able to potentiate gene expression programs associated with multiple cardiovascular lineages in CPCs, suggesting that GATA4 may be effective in priming CPCs for enhanced differentiation in the setting of stem cell therapy.

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways.

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit.

c-kit+ Cardiac stem cells alleviate post-myocardial infarction left ventricular dysfunction despite poor engraftment and negligible retention in the recipient heart.

Although transplantation of c-kit+ cardiac stem cells (CSCs) has been shown to alleviate left ventricular (LV) dysfunction induced by myocardial infarction (MI), the number of exogenous CSCs remaining in the recipient heart following transplantation and their mechanism of action remain unclear. We have previously developed a highly sensitive and accurate method to quantify the absolute number of male murine CSCs in female recipient organs after transplantation. In the present study, we used this method to monitor the number of donor CSCs in the recipient heart after intracoronary infusion. Female mice underwent a 60-min coronary occlusion followed by reperfusion; 2 days later, 100,000 c-kit+/lin- syngeneic male mouse CSCs were infused intracoronarily. Only 12.7% of the male CSCs present in the heart immediately (5 min) after infusion were still present in the heart at 24 h, and their number declined rapidly thereafter. By 35 days after infusion, only ∼ 1,000 male CSCs were found in the heart. Significant numbers of male CSCs were found in the lungs and kidneys, but only in the first 24 h. The number of CSCs in the lungs increased between 5 min and 24 h after infusion, indicating recirculation of CSCs initially retained in other organs. Despite the low retention and rapid disappearance of CSCs from the recipient heart, intracoronary delivery of CSCs significantly improved LV function at 35 days (Millar catheter). These results suggest that direct differentiation of CSCs alone cannot account for the beneficial effects of CSCs on LV function; therefore, paracrine effects must be the major mechanism. The demonstration that functional improvement is dissociated from survival of transplanted cells has major implications for our understanding of cell therapy. In addition, this new quantitative method of stem cell measurement will be useful in testing approaches of enhancing CSC engraftment and survival after transplantation.