Haeyoung Lim

Novel anticancer agent, benzyldihydroxyoctenone, isolated from Streptomyces sp. causes G1 cell cycle arrest and induces apoptosis of HeLa cells

In the course of screening for anticancer agents, a novel active compound, F3‐2‐5, was isolated from culture broth of Streptomyces sp., KACC91015. Its structure was identified using nuclear magnetic resonance, mass spectrometry, and molecular modeling experiments, and confirmed by total synthesis. The growth of various human cancer cell lines was inhibited in a dose‐dependent manner by 0.06–0.48 mM F3‐2‐5 over 24 h. Its IC50 values were estimated at 37 µM on HeLa, 72 µM on A549, and 190 µM on HT‐29 cells. However, F3‐2‐5 had no antiproliferative effect on normal lymphocytes and normal fibroblasts used as controls. Moreover, it affected cell cycle regulation and caused apoptosis of the HeLa cells; chromatin condensation and DNA fragmentation were observed in cells exposed to 80 µM F3‐2‐5. Western blot analysis revealed that F3‐2‐5 inhibited phosphorylation of retinoblastoma protein (pRb) and reduced expression of cyclin‐dependent kinase‐4 and ‐6, and cyclin D1 and E, while levels of p53 and p21WAF1/CIP1 increased. Taken together, these findings show that F3‐2‐5 inhibits proliferation of HeLa cells by inducing G1 phase arrest as a consequence of inhibition of pRb phosphorylation following up‐regulation of p21WAF1/CIP1 and p53. Furthermore, apoptosis in HeLa cells treated with F3‐2‐5 was associated with an increase in Bax and p53, leading to release of cytochrome c, activation of caspase‐3, and ‐8, and cleavage of poly (ADP‐ribose) polymerase. (Cancer Sci 2007; 98: 795–802)

HY253, a novel decahydrofluorene analog, from Aralia continentalis, induces cell cycle arrest at the G1 phase and cytochrome c-mediated apoptosis in human lung cancer A549 cells.

AIM OF THE STUDY In the course of our screening for novel modulators on cell cycle progression and apoptosis as anticancer drug candidates, we isolated a novel compound HY253 with the molecular structure of 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol from the roots of Aralia continentalis. This study was designed to evaluate the detailed mechanisms of cell cycle arrest and the apoptotic induction of HY253 in human lung cancer A549 cells. MATERIALS AND METHODS To investigate the effects of HY253 on cell cycle progression in A549 cells, we measured DNA content of A549 cells treated with 35 microM of HY253 using flow cytometric analysis. Furthermore, TUNEL assay was used to examine apoptotic induction in A549 cells treated with 70 microM of HY253 for 24 and 48 h. The effects of HY253 on apoptosis-associated and cell cycle regulatory proteins in A549 cells were examined using Western blot analysis. RESULTS The flow cytometric analysis revealed an appreciable G(1) phase arrest in A549 cells treated with 35 microM of HY253. This HY253-induced G(1) phase arrest is associated with decreased expression of cyclin D and up-regulation of p21(CIP1), via p53 phosphorylation at Ser-15, which resulted in increased hypophosphorylated pRb in A549 cells. Furthermore, TUNEL assay and Western blot analysis revealed an appreciable apoptotic induction in A549 cells treated with 70 microM of HY253 for 48 h. This apoptotic induction in HY253-treated A549 cells is also associated with cytochrome c release from mitochondria which in turn resulted in the activation of caspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). CONCLUSIONS These results demonstrate that HY253, a novel antiproliferative compound isolated from the roots of Aralia continentalis, induces cell cycle arrest at the G(1) phase and apoptosis in A549 cells. Based on these results, we suggest that HY253 may be a potent cancer chemotherapeutic candidate for use in treating human lung cancer cells via up-regulation and activation of p53 gene.

CR229, a novel derivative of β‐carbolin‐1‐one, induces cell cycle arrest and apoptosis in HeLa cells via p53 activation

In the course of screening for novel anticancer compounds, CR229 (6‐Bromo‐2,3,4,9‐tetrahydro‐carbolin‐1‐one), a novel derivative of β‐carbolin‐1‐one, was generated as a new scaffold candidate. For the first time, the authors demonstrate that CR229 inhibited the growth of HeLa cells by the induction of cell cycle arrest and apoptosis. Analysis of flow cytometry and western blots of HeLa cells treated with 2.5 µM CR229 revealed an appreciable cell cycle arrest in the G1, G2/M phase and apoptotic induction via the p53‐dependent pathway. Furthermore, the release of cytochrome c from mitochondria was detected using confocal microscopy in HeLa cells treated with CR229. Accordingly, these data demonstrate that the anticancer activity of CR229 is associated with: (i) the down‐regulation of cyclins and cyclin‐dependent kinase; (ii) the induction of p53, p21, and p16; and (iii) the activation of caspase‐3. (Cancer Sci 2007; 98: 1402–1407)

HY251, a novel cell cycle inhibitor isolated from Aralia continentalis, induces G1 phase arrest via p53-dependent pathway in HeLa cells.

This study was aimed to elucidate the novel structure of HY251 isolated from the roots of Aralia continentalis and to evaluate its detailed inhibition mechanisms on cell cycle progression in HeLa cells. The structure of HY251 was elucidated based on the interpretation of the NMR spectra, as 3-propyl-2-vinyl-1,2,3,3a,3b,6,7,7a,8,8a-decahydrocyclopenta[a]indene-3,3a,7a,8a-tetraol. The flow cytometric analysis revealed an appreciable G(1) phase arrest in HeLa cells treated with 100 microM of HY251. This HY251-induced G(1) phase arrest is associated with decreased expression of cyclin D3 and up-regulation of p21(CIP1) and p27(KIP1), via p53 phosphorylation at Ser-15 by transcriptional up-regulation of ATM, which resulted in increased hypophosphorylated pRb in HeLa cells.

HY253, a novel compound isolated from Aralia continentalis, induces apoptosis via cytochrome c-mediated intrinsic pathway in HeLa cells

This study was aimed to elucidate the novel structure of HY253 isolated from the roots of Aralia continentalis and to evaluate its detailed mechanisms on apoptotic induction in HY253-treated HeLa cells. The structure of HY253 was elucidated based on the interpretation of the NMR spectra, as 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol. The TUNEL assay using flow cytometer revealed an appreciable apoptotic induction in HeLa cells treated with 100 microM of HY253 for 48 h. This apoptotic induction is associated with cytochrome c release from mitochondria, via up-regulation of pro-apoptotic Bcl-2 proteins, such as Bax and Bak, which, in turn, resulted in the activation of caspase-8, -9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP).

Benzyldihydroxyoctenone, a novel anticancer agent, induces apoptosis via mitochondrial-mediated pathway in androgen-sensitive LNCaP prostate cancer cells

This study was aimed to evaluate detailed mechanisms on the apoptotic induction of benzyldihydroxyoctenone, a novel compound isolated from Streptomyces sp. KACC91015, in androgen-sensitive LNCaP prostate cancer cells. Benzyldihydroxyoctenone, designated as F3-2-5 in the current study, caused accumulation of apoptotic sub-G(1) phase in the flow cytometric analysis using propidium iodide staining. Moreover, the typical apoptotic DNA fragmentation of the LNCaP cells treated with 30 microM of F3-2-5 was confirmed using the TUNEL assay. This apoptotic induction of F3-2-5 in the LNCaP cells was associated with the cytochrome c release from mitochondria to cytosol, and the activation of procaspase-8, -9, and -3, as well as the specific proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). In addition, F3-2-5 treatment caused the down-regulation of the antiapoptotic protein, such as Bcl-2 and Bcl-X(L), but the proapoptotic protein, such as Bax, was not influenced. To investigate whether apoptotic induction by F3-2-5 is also due to the down-regulation of androgen receptor (AR), Western blot analysis and quantitative RT-PCR were conducted in F3-2-5-treated LNCaP prostate cancer cells. We found that F3-2-5 significantly inhibited the expression levels of AR and prostate-specific antigen (PSA) proteins in a time-dependent manner, as well as F3-2-5 abrogated the up-regulation of AR and PSA genes with and without DHT. Therefore, F3-2-5 has been shown to be an androgen antagonist, suggesting that F3-2-5 could be a potent agent for the treatment of both androgen-dependent and hormone-refractory prostate cancer.

Mononuclear cells from the cord blood and granulocytecolony stimulating factor-mobilized peripheral blood: is there a potential for treatment of cerebral palsy?

To investigate a possible therapeutic mechanism of cell therapy in the field of cerebral palsy using granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (mPBMCs), we compared the expression of inflammatory cytokines and neurotrophic factors in PBMCs and mPBMCs from children with cerebral palsy to those from healthy adult donors and to cord blood mononuclear cells donated from healthy newborns. No significant differences in expression of neurotrophic factors were found between PBMCs and mPBMCs. However, in cerebral palsy children, the expression of interleukin-6 was significantly increased in mPBMCs as compared to PBMCs, and the expression of interleukin-3 was significantly decreased in mPBMCs as compared to PBMCs. In healthy adults, the expression levels of both interleukin-1β and interleukin-6 were significantly increased in mPBMCs as compared to PBMCs. The expression of brain-derived neurotrophic factors in mPBMC from cerebral palsy children was significantly higher than that in the cord blood or mPBMCs from healthy adults. The expression of G-CSF in mPBMCs from cerebral palsy children was comparable to that in the cord blood but significantly higher than that in mPBMCs from healthy adults. Lower expression of pro-inflammatory cytokines (interleukin-1β, interleukin-3, and -6) and higher expression of anti-inflammatory cytokines (interleukin-8 and interleukin-9) were observed from the cord blood and mPBMCs from cerebral palsy children rather than from healthy adults. These findings indicate that mPBMCs from cerebral palsy and cord blood mononuclear cells from healthy newborns have the potential to become seed cells for treatment of cerebral palsy.