A novel BH3 mimetic S1 potently induces Bax/Bak‐dependent apoptosis by targeting both Bcl‐2 and Mcl‐1

Broad spectrum Bcl‐2 small molecule inhibitors act as BH3 mimetics are effective antitumor agents. Herein, we have identified S1 , a previously discovered small molecule Bcl‐2 inhibitor, as the first authentic BH3 mimetic as well as a dual, nanomolar inhibitor of Bcl‐2 and Mcl‐1 (K_i = 310 nM and 58 nM, respectively). The results of fluorescence polarization assays, coimmunoprecipitation, fluorescent resonance energy transfer, and shRNA indicated that S1 can disrupt Bcl‐2/Bax, Mcl‐1/Bak and Bcl‐2/Bim heterodimerization in multiple cell lines, activate Bax accompanied by its translocation to mitochondrial, activate caspase 3 completely dependent on Bax/Bak, and in turn induce a Bim‐independent apoptosis. Moreover, S1 could induce apoptosis on the primary acute lymphoblastic leukemia cells regardless of Mcl‐1 level. Mechanism‐based single agent antitumor activity in a mouse xenograft H22 (mouse liver carcinoma) model ascertain its therapeutic potential. S1 represents a novel chemical class of antitumor leads that function solely as BH3 mimetics and pan‐Bcl‐2 inhibitors. In the meanwhile, S1 could become a unique tool for interactions between Bcl‐2 family proteins.     

Inhibition of matrix metalloproteinase‐2 enhances radiosensitivity by abrogating radiation‐induced FoxM1‐mediated G2/M arrest in A549 lung cancer cells

Matrix metalloproteinase‐2 (MMP‐2), is known to degrade the collagen IV, plays a role in radiation‐induced lung injury. We therefore investigated the antitumor effects of combining MMP‐2 inhibition using an adenovirus expressing siRNA against MMP‐2 (Ad‐MMP‐2‐Si) with radiation therapy (IR) on A549 lung cancer cells in vitro and in vivo. IR increased MMP‐2 mRNA, protein and activity in lung cancer cells. MMP‐2 inhibition along with IR enhanced radiosensitivity as determined by clonogenic assay, flow cytometry and TUNEL assay. We show that MMP‐2 inhibition prior to irradiation reduced p53 phosphorylation, with a corresponding reduction in the expression of the p53 downstream target gene p21^Cip1/Waf1. Irradiated tumor cells induced the FoxM1‐mediated DNA repair gene, XRCC1 and Checkpoint kinases 2/1, which were abrogated with combined treatment of Ad‐MMP‐2‐Si and IR. Further, the combination of Ad‐MMP‐2‐Si with radiotherapy significantly increased antitumor efficacy in vivo compared to either agent alone. Indeed, histological analysis of tumor sections collected from the combination group revealed more apoptotic cells. These studies suggest that MMP‐2 inhibition in combination with radiotherapy abrogates G2 cell cycle arrest leading to apoptosis and provide evidence of the antitumor efficacy of combining MMP‐2 inhibition with irradiation as a new therapeutic strategy for the effective treatment of NSCLC patients. © 2008 Wiley‐Liss, Inc.     

p14ARF induces apoptosis via an entirely caspase‐3‐dependent mitochondrial amplification loop

The p14^ARF tumor suppressor triggers cell death or cell cycle arrest upon oncogenic stress. In MCF‐7 breast carcinoma cells, expression of the tumor suppressor gene p14^ARF fails to trigger apoptosis but induces an arrest in the G1 and, to a lesser extent, in the G2 phase in the cell division cycle. Here, inhibition of cell cycle arrest resulted in apoptosis induction in caspase‐3 proficient MCF‐7 cells upon expression of p14^ARF. This occurred in the absence of S‐phase progression or mitotic entry. In contrast, syngeneic, caspase‐3‐deficient MCF‐7 cells remained entirely resistant to p14^ARF‐induced apoptosis. Thus, cell cycle checkpoint abrogation overcomes resistance to p14^ARF‐induced cell death and promotes cell death via a caspase‐3‐dependent pathway. Cell death coincided with dissipation of the mitochondrial membrane potential, release of cytochrome c, and was inhibitable by pan‐caspase inhibitors and the caspase‐3/7 inhibitor zDEVD‐fmk. Of note, mitochondrial events of apoptosis execution depended entirely on caspase‐3 proficiency indicating that caspase‐3 either acts “up‐stream” of the mitochondria in a “non‐canonical” pathway or mediates a mitochondrial feedback loop to amplify the apoptotic caspase signal in p14^ARF‐induced stress signaling.     

The dual PI3K/mTOR inhibitor NVP‐BEZ235 and chloroquine synergize to trigger apoptosis via mitochondrial‐lysosomal cross‐talk

On the basis of our previous identification of aberrant phosphatidylinositol‐3‐kinase (PI3K)/Akt signaling as a novel poor prognostic factor in neuroblastoma, we evaluated the dual PI3K/mTOR inhibitor BEZ235 in the present study. Here, BEZ235 acts in concert with the lysosomotropic agent chloroquine (CQ) to trigger apoptosis in neuroblastoma cells in a synergistic manner, as calculated by combination index (CI < 0.5). Surprisingly, inhibition of BEZ235‐induced autophagy is unlikely the primary mechanism of this synergism as reported in other cancers, since neither inhibition of autophagosome formation by knockdown of Atg7 or Atg5 nor disruption of the autophagic flux by Bafilomycin A1 (BafA1) enhance BEZ235‐induced apoptosis. BEZ235 stimulates enlargement of the lysosomal compartment and generation of reactive oxygen species (ROS), while CQ promotes lysosomal membrane permeabilization (LMP). In combination, BEZ235 and CQ cooperate to trigger LMP, Bax activation, loss of mitochondrial membrane potential (MMP) and caspase‐dependent apoptosis. Lysosome‐mediated apoptosis occurs in a ROS‐dependent manner, as ROS scavengers significantly reduce BEZ235/CQ‐induced loss of MMP, LMP and apoptosis. There is a mitochondrial‐lysosomal cross‐talk, since lysosomal enzyme inhibitors significantly decrease BEZ235‐ and CQ‐induced drop of MMP and apoptosis. In conclusion, BEZ235 and CQ act in concert to trigger LMP and lysosome‐mediated apoptosis via a mitochondrial‐lysosomal cross‐talk. These findings have important implications for the rational development of PI3K/mTOR inhibitor‐based combination therapies.     

Inhibition of cyclin E1 overcomes temozolomide resistance in glioblastoma by Mcl‐1 degradation

Glioblastoma (GBM) is one of the major causes of brain cancer‐related mortality worldwide. Temozolomide (TMZ) is an important agent against GBM. Acquired TMZ‐resistance severely limits the chemotherapeutic effect and leads to poor GBM patient survival. To study the underlying mechanism of drug resistance, two TMZ resistant GBM cell lines, A172 and U87, were generated. In this study, the TMZ resistant cells have less apoptosis and cell‐cycle change in response to the TMZ treatment. Western blot results revealed that cyclin E1 was upregulation in TMZ resistant cells. Inhibition or depletion of cyclin E1 re‐sensitized the resistant cells to the TMZ treatment, which indicated the induction of cyclin E1 is the cause of TMZ resistance in GBM cells. Furthermore, we also found the expression of cyclin E1 stabilized the expression of Mcl‐1, which contributes to the TMZ resistance in GBM cells. Finally, our in vivo xenograft data showed that the combination of flavopiridol, a cyclin E1/CDK2 inhibitor, overcomes the TMZ resistant by inducing higher apoptosis. Overall, our data provided a rationale to overcome the TMZ resistant in GBM treatment by inhibiting the cyclin E1 activity.     

Antitumor activity of an anti‐CD98 antibody

CD98 is expressed on several tissue types and specifically upregulated on fast‐cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98‐specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell‐line derived xenograft models and was as efficacious as standard of care carboplatin in patient‐derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase‐3 and ‐7‐mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.     

Synergistic antitumor activity of the SN‐38‐incorporating polymeric micelles NK012 with S‐1 in a mouse model of non‐small cell lung cancer

The combination therapy of CPT‐11, a prodrug of SN‐38, with S‐1, a dihydropyrimidine dehydrogenase inhibitory fluoropyrimidine, shows a high clinical response rate in non‐small cell lung cancer (NSCLC). However, this combination causes severe toxicities such as diarrhea. Here, we investigated the advantages of treatment with the SN‐38‐incorporating polymeric micelles NK012 over CPT‐11 in combination with S‐1 in mice bearing a NSCLC xenograft in terms of antitumor activity and toxic effects, particularly intestinal toxicity. In vitro cytotoxic effects were examined in human NSCLC cell lines (A549, PC‐9, PC‐14, EBC‐1 and H520). In vivo antitumor effects were evaluated in PC‐14‐ and EBC‐1‐bearing mice after NK012 or CPT‐11 administration on Days 0 and 7 and S‐1 administration on Days 0–13. Pathological changes in the small intestine were also investigated. The in vitro growth inhibitory effects of NK012 were 56.8‐ to 622‐fold more potent than those of CPT‐11. NK012/S‐1 treatment showed significantly higher antitumor activity both in PC‐14‐bearing (p = 0.0007) and EBC‐1‐bearing mice (p < 0.0001) than CPT‐11/S‐1 treatment. The deformity and decrease in the density of intestinal villi were more severe in CPT‐11/S‐1‐treated mice than in NK012/S‐1‐treated mice. NK012/S‐1 combination is a promising candidate regimen against NSCLC without inducing toxicities such as severe diarrhea and therefore warrants clinical evaluation.     

Ursolic acid induces PC‐3 cell apoptosis via activation of JNK and inhibition of Akt pathways in vitro

Ursolic acid (UA), a pentacyclic triterpenoid compound, has been demonstrated to have an antiproliferative effect in various tumors. We investigated the cell killing effects of UA in the human hormone refractory prostate cancer cell line, PC‐3 cells. Also, the molecular mechanisms underlying its antigrowth effect were explored. We found that UA treatment in vitro can effectively inhibit PC‐3 cell viability in a dose‐dependent manner by inducing apoptosis, demonstrated by annexin V‐FITC/propidium iodide staining. Both extrinsic and intrinsic apoptotic pathways appear to be triggered by UA treatment, because inhibiting activation of both caspase‐8 and ‐9 could prevent UA‐induced apoptosis in PC‐3 cells. The c‐Jun N‐terminal kinase (JNK) was found to be activated, followed by Bcl‐2 phosphorylation and activation of caspase‐9. On the other hand, UA inhibited the Akt pathway, subsequently upregulating the expression of Fas ligand (FasL), which initiates death receptor‐mediated apoptosis in PC‐3 cells. Importantly, experimentally lowering FasL expression by siRNA significantly inhibited UA‐induced caspase‐8 activation and at least partly attenuated the consequent apoptosis, suggesting an involvement of FasL and its regulating pathway in the cell killing effect of UA. UA also inhibited cell invasion by downregulating matrix metalloproteinase‐9 via inhibition of Akt in PC‐3 cells. Although further evaluation of the UA effects in vivo is needed, the present results suggest the potential utility of UA as a novel therapeutic agent in advanced prostate cancer. © 2010 Wiley‐Liss, Inc.     

Enhanced anti‐angiogenic effect of E7820 in combination with erlotinib in epidermal growth factor receptor–tyrosine kinase inhibitor‐resistant non‐small‐cell lung cancer xenograft models

Most non‐small‐cell lung cancers (NSCLCs) harboring activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to EGFR tyrosine kinase inhibitors (EGFR‐TKIs); however, they invariably develop resistance to these drugs. E7820 is an angiogenesis inhibitor that decreases integrin‐α2 expression and is currently undergoing clinical trials. We investigated whether E7820 in combination with erlotinib, an EGFR‐TKI, could overcome EGFR‐TKI‐resistance in the NSCLC cell lines A549 (KRAS; G12S), H1975 (EGFR; L858R/T790M), and H1650 (PTEN; loss, EGFR; exon 19 deletion), which are resistant to erlotinib. Immunohistochemical analysis was carried out in xenografted tumors to investigate anti‐angiogenesis activity and endothelial cell apoptosis levels by endothelial cell marker CD31 and TUNEL staining, respectively. Treatment with E7820 (50 mg/kg) with erlotinib (60 mg/kg) showed a synergistic antitumor effect in three xenograft models. Immunohistochemical analysis indicated that combined treatment with E7820 and erlotinib significantly decreased microvessel density and increased apoptosis of tumor‐associated endothelial cells compared with use of only one of the agents. This combination increased apoptosis in HUVECs through activation of both intrinsic and extrinsic apoptosis pathways in vitro. The combination of E7820 with erlotinib is an alternative strategy to overcome erlotinib resistance in NSCLC by enhancement of the anti‐angiogenic activity of E7820.     

Antitumor activity and pharmacologic characterization of the depsipeptide analog as a novel histone deacetylase/ phosphatidylinositol 3‐kinase dual inhibitor

Histone deacetylase (HDAC)/phosphatidylinositol 3‐kinase (PI3K) dual inhibition is a promising strategy for the treatment of intractable cancers because of the advantages of overcoming potential resistance and showing synergistic effects. Therefore, development of an HDAC/PI3K dual inhibitor is reasonably attractive. Romidepsin (FK228, depsipeptide) is a potent HDAC inhibitor. We previously reported that depsipeptide and its analogs have an additional activity as PI3K inhibitors and are defined as HDAC/PI3K dual inhibitors. Subsequently, we identified FK‐A11 as the most potent analog and reported its biochemical, biological, and structural properties as an HDAC/PI3K dual inhibitor. In this study, we reveal the in vitro and in vivo efficacy of FK‐A11 in HT1080 fibrosarcoma and PC3 prostate cancer cell xenograft mouse models. FK‐A11 showed in vivo antitumor activity by both i.v. and i.p. administration in a dose‐dependent manner. In both xenograft models, FK‐A11 showed superior antitumor effects compared to other depsipeptide analogs in accordance with in vitro anti‐cell proliferation effects and the potency of HDAC/PI3K dual inhibition. In addition, we showed evidence of HDAC/PI3K dual inhibition accompanying antitumor efficacy in xenograft tumor tissues by immunohistochemistry. We also detailed pharmacokinetic characterization of FK‐A11 in mice. These findings will be essential for guiding further preclinical and clinical studies.     

Pemetrexed induces both intrinsic and extrinsic apoptosis through ataxia telangiectasia mutated/p53‐dependent and ‐independent signaling pathways

Pemetrexed, a new‐generation antifolate, has demonstrated promising single‐agent activity in front‐ and second‐line treatments of non‐small cell lung cancer. However, the molecular mechanism of pemetrexed‐mediated antitumor activity remains unclear. The current study shows that pemetrexed induced DNA damage and caspase‐2, ‐3, ‐8, and ‐9 activation in A549 cells and that treatment with caspase inhibitors significantly abolished cell death, suggesting a caspase‐dependent apoptotic mechanism. The molecular events of pemetrexed‐mediated apoptosis was associated with the activation of ataxia telangiectasia mutated (ATM)/p53‐dependent and ‐independent signaling pathways, which promoted intrinsic and extrinsic apoptosis by upregulating Bax, PUMA, Fas, DR4, and DR5 and activating the caspase signaling cascade. Supplementation with dTTP allowed normal S‐phase progression and rescued apoptotic death in response to pemetrexed. Overall, our findings reveal that the decrease of thymidylate synthase and the increase of Bax, PUMA, Fas, DR4, and DR5 genes may serve as biomarkers for predicting responsiveness to pemetrexed. © 2011 Wiley Periodicals, Inc.     

MicroRNA‐155‐5p promotes hepatocellular carcinoma progression by suppressing PTEN through the PI3K/Akt pathway

MicroRNA‐155‐5p (miR‐155‐5p) has been reported to play an oncogenic role in different human malignancies; however, its role in hepatocellular carcinoma (HCC) progression is not clearly understood. In this study, we used real‐time PCR in 20 rats with chemically‐induced HCC, 28 human HCC tissues, and the matched paracarcinoma tissues, and HCC cell lines to determine the expression patterns of miR‐155‐5p and PTEN mRNA. Algorithm‐based and experimental strategies, such as dual luciferase gene reporter assays, real‐time PCR and western blots were used to identify PTEN as a candidate miR‐155‐5p target. Gain‐ and loss‐of‐function experiments and administration of a PI3K/Akt pathway inhibitor (wortmannin) were used to identify the effects of miR‐155‐5p and PTEN in MTT assays, flow cytometric analysis, wound healing assays and transwell assays. The results showed that miR‐155‐5p was highly overexpressed; however, PTEN was underexpressed in the HCC rat models, human HCC tissues and cell lines. In addition, miR‐155‐5p upregulation and PTEN downregulation were significantly associated with TNM stage (P < 0.05). Through in vitro experiments, we found that miR‐155‐5p promoted proliferation, invasion and migration, but inhibited apoptosis in HCC by directly targeting the 3′‐UTR of PTEN. Western blots showed that miR‐155‐5p inactivated Bax and caspase‐9, but activated Bcl‐2 to inhibit apoptosis, and it activated MMP to promote migration and invasion via the PI3K/Akt pathway. A xenograft tumor model was used to demonstrate that miR‐155‐5p targets PTEN and activates the PI3K/Akt pathway in vivo as well. Our study highlighted the importance of miR‐155‐5p and PTEN associated with aggressive HCC both in vitro and in vivo.     

μ‐Calpain regulates caspase‐dependent and apoptosis inducing factor‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis

Cisplatin, an effective anticancer agent, can induce tumor cell apoptosis via caspase‐dependent and‐independent pathways. However, the precise mechanism that regulates the pathways remains unclear. In this study, we showed that μ‐calpain mediated both caspase‐dependent and‐independent pathways during cisplatin‐induced apoptosis in human lung adenocarcinoma cells. After cisplatin treatment, calpain activation, as measured by a fluorescent substrate, was an early event, taking place well before apoptosis inducing factor (AIF) release and caspase‐9/‐3 activation. Confocal imaging of cells transfected with AIF‐GFP demonstrated that AIF release occurred about 9 hr after cisplatin treatment. The increase of μ‐calpain activity proved to be a crucial event in the apoptotic machinery, as demonstrated by the significant protection of cell death in samples suppressed the endogenous μ‐calpain expression level, as well as cotreated with the calpain inhibitors, calpeptin and PD150606. Inhibition of μ‐calpain not only significantly reduced caspase‐9/‐3 activities but also completely blocked AIF redistribution. Our study also showed that endogenous mitochondrial μ‐calpain could directly induce the truncation and release of AIF, while caspases and cathepsins were not necessary for this process. In conclusion, the study demonstrated that activation of μ‐calpain played an essential role in regulating both caspase‐dependent and AIF‐mediated caspase‐independent apoptotic pathways in cisplatin‐induced apoptosis. © 2009 UICC     

SYUNZ‐16, a newly synthesized alkannin derivative,induces tumor cells apoptosis and suppresses tumor growth through inhibition of PKB/AKT kinase activity and blockade of AKT/FOXO signal pathway

Alkannin is the major bioactive compound of Arnebia euchroma roots, which is used in many therapeutic remedies in Chinese traditional medicine. SYUNZ‐16 is a new derivative of alkannin. In this study, anticancer effects of SYUNZ‐16 on human lung adenocarcinoma cell line GLC‐82 and human hepatocarcinoma cell line Hep3B were tested in vitro. The results showed SYUNZ‐16 could obviously inhibit the proliferation of these cancer cell lines via induction of apoptosis, with the evidence of increasing AnnexinV‐positive cells and cleaved caspase‐3 and PARP fragments. More importantly, we found that SYUNZ‐16 could inhibit AKT activity in cell‐free system. Treatment of cancer cells with SYUNZ‐16 decreased the phosphorylation of AKT. Additionally, SYUNZ‐16 partially attenuated the phosphorylation levels of FKHR and FKHRL1 in a dose‐dependent and time‐dependent fashion, and led to an increase in the nuclear accumulation of exogenous FKHR, and upregulated the mRNA expression of Bim and TRADD in cancer cells. Further study showed that constitutively activated AKT1 transfection could reduce apoptosis induction mediated by SYUNZ‐16. The in vivo experiments showed that SYUNZ‐16 had inhibitory effects on S‐180 sarcoma implanted to mice. And in GLC‐82 xenograft models, SYUNZ‐16 at 20 mg/kg/qod remarkably inhibited the tumor growth with the T/C value of 45.3%. Taken together, SYUNZ‐16 might be a potent inhibitor of AKT signaling pathway in tumor cells. These data provide evidence for the development of SYUNZ‐16 as a potential antitumor drug candidate for further research and development.     

Antitumor effects of novel compound,guttiferone K,on colon cancer by p21Waf1/Cip1‐mediated G0/G1 cell cycle arrest and apoptosis

Low selectivity is one of the major problems of currently used anticancer drugs, therefore, there is a high demand for novel, selective antitumor agents. In this study, the anticancer effects and mechanisms of guttiferone K (GUTK), a novel polyprenylated acylphloroglucinol derivative isolated from Garcinia cowa Roxb., were examined for its development as a novel drug targeting colon cancer. GUTK concentration‐ and time‐dependently reduced the viability of human colon cancer HT‐29 cells (IC₅₀ value 5.39 ± 0.22 μM) without affecting the viability of normal human colon epithelial CCD 841 CoN cells and induced G₀/G₁ cell cycle arrest in HT‐29 cells by down‐regulating cyclins D1, D3 and cyclin‐dependent kinases 4 and 6, while selectively restoring p21Waf1/Cip1 and p27Kip1 to levels comparable to those observed in normal colon cells, without affecting their levels in normal cells. GUTK (10.0 μM) induced cleavage of PARP, caspases‐3, ‐8 and ‐9 and chromatin condensation to stimulate caspase‐3‐mediated apoptosis. The addition of a JNK inhibitor, SP600125, partially reversed GUTK‐induced caspase‐3 activity, indicating the possible involvement of JNK in GUTK‐induced apoptosis. Furthermore, GUTK (10 mg/kg, i.p.) significantly decreased the tumor volume in a syngeneic colon tumor model when used alone or in combination with 5‐fluorouracil without toxicity to the mice. Immunohistochemical staining of the tumor sections revealed a mechanism involving an increase in cleaved caspase‐3 and a decrease in cell proliferation marker Ki‐67. Our results support GUTK as a promising novel, potent and selective antitumor drug candidate for colon cancer.     

20(S)‐ginsenoside‐Rg3 reverses temozolomide resistance and restrains epithelial‐mesenchymal transition progression in glioblastoma

Glioblastoma multiforme (GBM) is one of the most malignant human intracranial tumors. Temozolomide (TMZ) is the primary alkylating agent for GBM patients. However, many GBM patients are resistant to TMZ. Therefore, patients with GBM urgently need more effective therapeutic options. 20(S)‐ginsenoside‐Rg3 (20(S)‐Rg3) is a natural chemical with anti‐tumor effects, but at present there is little understanding of its functional mechanism. Several research reports have demonstrated that O⁶‐methylguanine DNA‐methyltransferase (MGMT) repairs damaged DNA and contributes to TMZ resistance in gliomas. In addition, recent studies have shown that MGMT gene expression could be regulated by the Wnt/β‐catenin pathway. However, whether 20(S)‐Rg3 inhibits MGMT expression and augments chemosensitivity to Temozolomide (TMZ) in glioma cells remains unclear. In this study, we explored the modulating effects of 20(S)‐Rg3 on MGMT. We used glioma cell lines, primary cell strain (including T98G, U118 and GBM‐XX; all of them are MGMT‐positive glioma cell lines) and xenograft glioma models to examine whether 20(S)‐Rg3 increased the sensitivity to TMZ and to reveal the underlying mechanisms. We found that the MGMT expression was effectively downregulated by 20(S)‐Rg3 via the Wnt/β‐catenin pathway in glioma cell lines, and TMZ resistance was significantly reversed by 20(S)‐Rg3. Meanwhile, 20(S)‐Rg3 shows no obvious cytotoxicity at its effective dose and is well tolerated in vivo. In addition, we found that 20(S)‐Rg3 significantly restrains the epithelial‐mesenchymal transition (EMT) progression of glioma cells. Taken together, these results indicate that 20(S)‐Rg3 may be a novel agent to use in treatment of GBM, especially in TMZ‐resistant GBM with high MGMT expression.     

Apoptotic signaling in bufalin‐ and cinobufagin‐treated androgen‐dependent and ‐independent human prostate cancer cells

Prostate cancer has its highest incidence in the USA and is becoming a major concern in Asian countries. Bufadienolides are extracts of toxic glands from toads and are used as anticancer agents, mainly on leukemia cells. In the present study, the antiproliferative and apoptotic mechanisms of bufalin and cinobufagin on prostate cancer cells were investigated. Proliferation of LNCaP, DU145, and PC3 cells was measured by 3‐(4,5‐dimethylthiazol‐2‐yle)‐2,5‐diphenyltetrazolium bromide assay and the doubling time (tD) was calculated. Bufalin and cinobufagin caused changes in the tD of three prostate cancer cell lines, which were more significant than that of human mesangial cells. In addition, bufadienolides induced prostate cancer cell apoptosis more significantly than that in breast epithelial cell lines. After treatment, the caspase‐3 activity and protein expression of caspase‐3, ‐8, and ‐9 were elevated. The expression of other apoptotic modulators, including mitochondrial Bax and cytosolic cytochrome c, were also increased. However, expression of p53 was only enhanced in LNCaP cells. Downregulation of p53 by antisense TP53 restored the cell viability suppressed by bufalienolides. Furthermore, the increased expression of Fas was more significant in DU145 and PC3 cells with mutant p53 than in LNCaP cells. Transfection of Fas small interfering RNA restored cell viability in the bufadienolide‐treated cells. These results suggest that bufalin and cinobufagin suppress cell proliferation and cause apoptosis in prostate cancer cells via a sequence of apoptotic modulators, including Bax, cytochrome c, and caspases. The upstream mediators might be p53 and Fas in androgen‐dependent LNCaP cells and Fas in androgen‐independent DU145 and PC3 cells. (Cancer Sci 2008; 99: 2467–2476)