Cell Cycle Distribution Assays Research Articles

Anti-proliferative of physcion 8-O-β-glucopyranoside isolated from Rumex japonicus Houtt. on A549 cell lines via inducing apoptosis and cell cycle arrest.

BackgroundLung cancers are leading causes of cancer death, and Rumex japonicus has been traditionally used in folk medicine as anti-microorganic, anti-inflammatory and anti-tumor agents. This study was designed to investigate the anti-proliferative activity of physcion 8-O-β-glucopyranoside (PG) isolated from Rumex japonicus Houtt. on A549 cell lines.MethodsIn our present study, PG was isolated and identified from the ethanol extracts of R. japonicus. MTT method was used to evaluate the anti-proliferative activity of PG on A549 cell lines, and cell cycle distribution assay, apoptosis assay, and western blot analysis in vitro were used to explore the possible mechanisms.ResultsFrom the results of our present study, cell viability was obviously inhibited by PG, in a dose- and time-dependent manner. Our results also suggested that the anti-proliferative effect of PG was related to cell cycle arrest at the G2/M phase through repression of cdc2 and Cyclin B1 protein expression. In addition, the results of apoptosis assay and western blot analysis indicated that the anti-proliferative activity could be related to apoptosis via up-regulating the expressions of Bax, caspase-3 and caspase-7, and down-regulating the expressions of Bcl-2.ConclusionsIn conclusion, the PG has significant anti-proliferative activity on A549 cell lines, and the possible mechanism was related to cell cycle arrest at the G2/M phase, and apoptosis via the regulations of Bax, Bcl-2, and caspase-3 and caspase-7.

Altered microRNA expression profiles are involved in resistance to low-dose ionizing radiation in the absence of BMI1 in human dermal fibroblasts.

The polycomb group RING finger protein, B-cell‑specific moloney murine leukemia virus integration site1 (BMI1), has emerged as a key regulator of cell proliferation, cell cycle, cell immortalization, chemoresistance and radioresistance. Although the radioresistant effect of BMI1 has been thoroughly investigated, the effectiveness of this factor on low-dose radiation (LDR) resistance has not been explored. Here, we demonstrate that BMI1 is not critical for altering cell viability or cell growth in response to LDR, but BMI1 changes cellular gene expression profiles in response to LDR. Normal human dermal fibroblasts (NHDFs) stably expressing BMI1 short hairpin RNA (shRNA) did not exhibit changes in cell viability or cell cycle distribution assays following exposure to 0.1Gy of γ-radiation. However, microRNA (miRNA) microarrays revealed that a lack of BMI1 leads to changes in miRNA expression in response to LDR. Bioinformatics analyses demonstrated that predicted target genes of the altered miRNAs are functionally involved in both negative and positive regulation of cell growth, cell proliferation, cell cycle and apoptosis. Therefore, these results indicate that low radiosensitivity even in the absence of the radioresistant factor BMI1 is related with the altered miRNA expression profiles in NHDF.

PDGF receptor alpha inhibition induces apoptosis in glioblastoma cancer stem cells refractory to anti-Notch and anti-EGFR treatment.

BackgroundCancer stem cells (CSC) represent a rare fraction of cancer cells characterized by resistance to chemotherapy and radiation, therefore nowadays there is great need to develop new targeted therapies for brain tumors and our study aim to target pivotal transmembrane receptors such as Notch, EGFR and PDGFR, which are already under investigation in clinical trials setting for the treatment of Glioblastoma Multiforme (GBM).MethodsMTS assay was performed to evaluate cells response to pharmacological treatments. Quantitative RT-PCR and Western blots were performed to state the expression of Notch1, EGFR and PDGFRα/β and the biological effects exerted by either single or combined targeted therapy in GBM CSC. GBM CSC invasive ability was tested in vitro in absence or presence of Notch and/or EGFR signaling inhibitors.ResultsIn this study, we investigated gene expression and function of Notch1, EGFR and PDGFR to determine their role among GBM tumor core- (c-CSC) vs. peritumor tissue-derived cancer stem cells (p-CSC) of six cases of GBM. Notch inhibition significantly impaired cell growth of c-CSC compared to p-CSC pools, with no effects observed in cell cycle distribution, apoptosis and cell invasion assays. Instead, anti-EGFR therapy induced cell cycle arrest, sometimes associated with apoptosis and reduction of cell invasiveness in GBM CSC. In two cases, c-CSC pools were more sensitive to simultaneous anti-Notch and anti-EGFR treatment than either therapy alone compared to p-CSC, which were mostly resistant to treatment. We reported the overexpression of PDGFRα and its up-regulation following anti-EGFR therapy in GBM p-CSC compared to c-CSC. RNA interference of PDGFRα significantly reduced cell proliferation rate of p-CSC, while its pharmacological inhibition with Crenolanib impaired survival of both CSC pools, whose effects in combination with EGFR inhibition were maximized.ConclusionsWe have used different drugs combination to identify the more effective therapeutic targets for GBM CSC, particularly against GBM peritumor tissue-derived CSC, which are mostly resistant to treatments. Overall, our results provide the rationale for simultaneous targeting of EGFR and PDGFR, which would be beneficial in the treatment of GBM.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-247) contains supplementary material, which is available to authorized users.

Abstract B113: GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis.

Abstract Purpose of the study. GSK3A and GSK3B isoforms perform similar functions but they are not completely redundant, depending on the cell type, biological process and differentiative status (Rayasam GV et al, Br J Pharmacol, 2009). We recently demonstrated that GSK3B inhibition enables necroptosis in response to chemotherapy in drug-resistant colon carcinoma cells (Grassilli E et al, Clin Cancer Res, 2013). Here we investigated whether GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. Materials and Methods. Cell lines stably interfered were obtained by retroviral infection and selection with the appropriate antibiotic. siRNA transfection was performed using Lipofectamine 2000 (Invitrogen). Cell viability was assessed by Trypan blue staining. Cell proliferation was evaluated by CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega). Cell cycle was analysed by flow cytometry upon PI staining using Modfit Cell Cycle Analysis (Becton Dickinson). Reporter activity was analysed by Dual-Glo Luciferase Assay (Promega). IF staining was performed on 4% paraformaldehyde-fixed cells. Active caspase-3/7 was quantified by the Caspase-Glo3/7 Assay System (Promega). Western blots were performed on lysates obtained by RIPA buffer. Results and Discussion. GSK3A stable silencing in HCT116p53KO cells did not alter cell proliferation as assessed by growth curve, cell cycle distribution and β-catenin activation assays. GSK3A depletion abolished colony formation after 5FU treatment and re-sensitized drug-resistant HCT116p53KO cells to 5FU-induced cell death. Blocking GSK3A did not interfere with DNA damage sensing upon 5FU treatment, as demonstrated by H2AX foci formation, but impaired the DNA repair response by affecting RPA70 foci formation. GSK3A silencing allowed a cell death response to DNA-damaging drugs in absence of p53: 5FU-treated, GSK3A-depleted HCT116p53KO cells underwent caspase-independent PARP1- and tBid-dependent cell death, which was unaffected by RIP1 inhibition and was characterized by PARP-dependent nuclear re-localization of AIF. Our data demostrate that GSK3A, like GSK3B, contributes to cell survival upon DNA-damaging chemotherapy by suppressing RIP-independent necroptosis and pinpoint that inhibition of one isoform is sufficient to bypass drug resistance of p53-null cells. Being GSK3A and GSK3B redundant in the response to DNA-damaging drugs, it is likely that, when used together with anticancer drugs, GSK3 inhibitors should be particularly effective in overcoming drug resistance even at low doses, since inhibition of only one isoform, or rather partial inhibition of overall cellular GSK3 activity, is enough to re-sensitize drug-resistant cells. We therefore propose that GSK3 inhibition in combination with DNA-damaging drugs would be an appealing strategy to induce necroptosis in p53-null drug-resistant tumor. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B113. Citation Format: Emanuela Grassilli, Elena Federzoni, Robert Narloch, Leonarda Ianzano, Kristian Helin, Marialuisa Lavitrano. GSK3A is redundant with GSK3B in modulating drug resistance and chemotherapy-induced necroptosis. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B113.

Abstract 3664: Silibinin induces cell cycle arrest and apoptotic death via targeting hedgehog signaling in basal cell carcinoma cells.

Abstract Skin cancer is the most common malignancy in the United States with the number of incidences persistently rising due to the depletion of the ozone layer and lack of adequate solar protection. Non-melanoma skin cancer (NMSC), composed of basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), encompasses the vast majority of skin cancers with over 2 million new cases each year. Silibinin, an active bioflavanone was previously shown to have major protective effect against ultraviolet B radiation (UVB)-induced SCC through promoting DNA damage repair as well as inhibiting inflammation and angiogenesis. To date, however, the efficacy of silibinin has not been tested against BCC, which alone accounts for about 80% of NMSC incidences. BCC mostly causes disfigurement, pain and morbidity, and warrants greater attention for its prevention and treatment. The hallmark of BCC has been the constitutive activation of hedgehog (Hh) signaling pathway, mostly through the loss of function mutations in the patched (PTCH) gene. In the present study, we utilized ASZ001 cell line, derived from a mouse BCC tumor induced by UV-radiation in a PTCH1 heterozygous mouse, to evaluate the anti-cancer efficacy of silibinin against BCC. ASZ001 cells lack both copies of the PTCH1 gene and closely resembles the PTCH1 status in human BCC. We treated ASZ001 cells with either DMSO (control) or various doses of silibinin (25-100 μM), and performed trypan blue exclusion, clonogenic, apoptosis and cell cycle distribution assays. Results showed that silibinin treatment (25-100 μM) decreases the total cell number by 38-51% (p<0.001), 67-84% (p<0.001) and 40-95% (p<0.05-p<0.001) after 24, 48 and 72 h of treatment. Clonogenic assay further confirmed the decrease in cell viability by silibinin, and we observed 18% (p<0.05), 92% (p<0.001) and 100% (p<0.001) decrease in colony formation with 25, 50 and 100 μM doses of silibinin, respectively, in ASZ001 cells. Trypan blue exclusion assay showed that silibinin treatment (25-100 μM) also increased the cell death by 2 folds (p<0.05), 3 folds (p<0.001), and 4 folds (p<0.001) after 24, 48 and 72 h of treatment while annexin/PI staining confirmed that silibinin-induced cell death in ASZ001 cells is mainly apoptotic in nature. FACS analyses revealed that silibinin treatment significantly modulated the cell cycle distribution in ASZ001 cells, and we observed an S-phase arrest at 50 μM silibinin dose and a G2M arrest at 100 μM silibinin dose after 24 and 48 h of treatment. Molecular studies showed that silibinin treatment strongly decreases the expression of hedgehog signaling transcriptional factor Gli1, which is typically overexpressed in BCC and is known to regulate the expression of several oncogenes. Together, these results provide the first evidence of silibinin's efficacy against BCC cells, and suggest that silibinin could be useful in the prevention and/or treatment of both types of NMSCs. Citation Format: Cynthia Tilley, Gagan Deep, Chapla Agarwal, Rajesh Agarwal. Silibinin induces cell cycle arrest and apoptotic death via targeting hedgehog signaling in basal cell carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3664. doi:10.1158/1538-7445.AM2013-3664

Abstract 5478: A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2.

Abstract Esophageal cancer is one of the most common cancers, and the 5-year survival rate is less than 10% due to lack of effective therapeutic agents. This study was to evaluate antitumor activity of Ec-LDP-Hr-AE, a recently developed bispecific enediyne-energized fusion protein, on esophageal carcinoma, targeting both EGFR (epidermal growth factor receptor) and HER2 (human epidermal growth factor receptor 2), because both EGFR and HER2 have been shown overexpression in over 30% of esophageal cancers. The fusion protein Ec-LDP-Hr-AE was encoded by a gene that was genetically constructed by fusing the oligopeptides ligand Ec (22 amino acids of EGF COOH-terminal) and Hr (VH CDR3 region of anti-HER2 C6.5 antibody) with lidamycin (LDM, an enediyne antibiotic with potent cytotoxicity) via two (G4S)3-linker. The encoded 18.4 kDa fusion protein was expressed in Escherichia coli and characterized its binding affinity with esophageal squamous cell carcinoma (ESCC) cells by flow-cytometry-based immunofluorescence assay. The results showed that Ec-LDP-Hr-AE protein bound to ESCC cells EC9706 and KYSE150 with high affinity (the Kd values were 5.283μmol/L and 3.562μmol/L, respectively). The potency of Ec-LDP-Hr-AE was determined on four human ESCC cell lines with differential expression of EGFR and HER2 using MTT assay. The bispecific Ec-LDP-Hr-AE showed potent cytotoxicity to EC1, EC109, EC9706 and KYSE150 cells with IC50 values in the picomolar level. Moreover, the bispecific Ec-LDP-Hr-AE was more potent than the monospecific counterparts (Ec-LDP-AE and LDP-Hr-AE) and LDM. Cell cycle distribution assay revealed that Ec-LDP-Hr-AE caused G2-M arrest in EC9706 and KYSE150 cells, in which the G2-M phase cells reached the peak with 0.1nmol/L of Ec-LDP-Hr-AE treatment. Ec-LDP-Hr-AE also induced cancer cell apoptosis in EC9706 and KYSE150 cells and the induction of apoptosis was in a dosage-dependent manner. The apoptotic index in the EC9706 cells exposed to 0.1, 0.5, or 1 nmol/L of Ec-LDP-Hr-AE were 15.0 ± 0.3%, 38.1 ± 0.6%, and 50.0 ± 0.4%, respectively. Western blotting showed that Ec-LDP-Hr-AE promoted caspase-3 and caspase-7 activities as well as PARP cleavage, indicating that Ec-LDP-Hr-AE-induced apoptosis was associated with mitochondria-caspase cascade. Moreover, Ec-LDP-Hr-AE inhibited KYSE150 cancer cell proliferation via decreasing phosphorylation of EGFR and HER2, and further exerted inhibition of the activation of their downstream signaling molecules (e.g. AKT and ERK). In summary, the bispecific energized fusion protein Ec-LDP-Hr-AE exhibited potent cytotoxicity to ESCC cells in vitro, leading to cell cycle arrest and apoptosis via targeting EGFR and HER2. These results suggest that Ec-LDP-Hr-AE could be a promising candidate for esophageal cancer targeting therapy. Citation Format: Guo Xiaofang, Xiaofei Zhu, Yue Shang, Shenghua Zhang, Yongsu Zhen, Wancai Yang. A bispecific enediyne-energized fusion protein Ec-LDP-Hr-AE enhances anti-cancer efficacy on esophageal cancer cells by targeting EGFR and HER2. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5478. doi:10.1158/1538-7445.AM2013-5478

Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells

BackgroundThe high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined.MethodsThe levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H2O2, high glucose/U0126 or normal glucose/H2O2/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions.ResultsWe found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H2O2 stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor abolished the proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression under high glucose or normal glucose/H2O2 conditions.ConclusionsThese results demonstrate that the downstream effectors of Irf-1 are cyclin E/CDK2 during the high glucose-induced proliferation of VSMCs, whereas they are cyclin D1/CDK4 in normal glucose conditions. The Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression are associated with ROS/Erk1/2 signaling pathway under high glucose conditions.

A Copper Complex, ghn‐12, as a sensitization of DNA to UVA offers potential for a novel photochemotherapy

Photochemotherapy is a modern therapy modality, based upon the application of a photosensitizing agent onto the tissue followed by illumination with light sources, which is a proven therapeutic strategy for a number of non‐malignant hyperproliferative skin diseases and various cancers. The aim of the present study is to investigate a new synthesis copper complex (ghn‐12, Chemical Formula: C53H39ClCuN8O5) as a potent photocytotoxic agent. Photoinduced DNA cleavage activity, cell cytotoxicity, cell cycle distribution and apoptosis assay are used to investigate the effect of ghn‐12 on therapeutic applications. Ghn‐12 induced supercoiled pUC19 DNA cleavage from supercoiled to nicked circular form at a low‐power monochromatic UV‐A light of 365 nm. Flow cytometry analysis revealed that photoinduced ghn‐12 sensitized squamous cell carcinoma (SCC15) cells to arrest in the G0/G1 and S‐G2/M phases of the cell cycle with a concomitantly significant increase in the sub‐G1 cell population, indicating cell death by apoptosis. This photoinduced apoptosis process was accompanied by activation of caspase‐3 expression and FITC Annexin V staining after SCC15 cells treated with ghn‐12. Our data suggest that a new photosensitizing compound, ghn‐12, exhibiting light‐induced cleavage of double‐stranded DNA and causing apoptosis of squamous cell carcinoma. Ghn‐12 is a potential therapeutic reagent. Research support by grant from the National Science Council, Taiwan Grant NSC99‐2314‐B‐384 ‐002 ‐MY3.

Menthol Inhibits the Proliferation and Motility of Prostate Cancer DU145 Cells

In recent years, the transient receptor potential melastatin member 8 (TRPM8) channel has emerged as a promising prognostic marker and putative therapeutic target in prostate cancer. We have found that forced overexpression of TRPM8 in PC-3 cells can inhibit the cell proliferation and motility probably through the TRPM8 activation. In this study, we aimed to investigate whether activating the TRPM8 channel by its selective agonist menthol can inhibit the proliferation and motility of androgen-independent prostate cancer (AIPC) with remarkable expression of TRPM8. Menthol is a naturally occurring compound, which has been widely used in cosmetics and pharmaceutical products, and also as flavoring in food. DU145 cells are androgen-independent but have a remarkable expression of TRPM8. The demonstration of the existence of TRPM8 and the absence of TRPA1 in DU145 cells provided the foundation for the following experiments, because both TRPM8 and TRPA1 are molecular targets of menthol. The outcome of MTT assay indicated that menthol inhibited the cell growth (p < 0.01). Cell cycle distribution and scratch assay analysis revealed that menthol induced cell cycle arrest at the G(0)/G(1) phase (p < 0.01). Furthermore, menthol inhibited the migration of DU145 cells by downregulating the focal-adhesion kinase. So it suggests that the activation of the existing TRPM8 channels may serve as a potential and pragmatic treatment for those AIPC with remarkable expression of TRPM8, and menthol is a useful compound for future development as an anticancer agent.

Survivin-mediated Therapeutic Efficacy of Gemcitabine through Glucose-regulated Protein 78 in Hepatocellular Carcinoma

Survivin is an antiapoptotic molecule that is widely expressed in cancers, including hepatocellular carcinoma (HCC). Survivin has become a general therapeutic target for cancers because of its selective overexpression in a majority of tumors. However, little is known regarding the effect of survivin expression in combination with gemcitabine on HCC. We generated survivin knockdown cells (survivin-KD) via a short interfering RNA (siRNA) technique. The antiproliferation effects of gemcitabine were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assay, and cell cycle evaluation. According to the MTT assay, we found that survivin-KD cells were more sensitive than parental cells and scrambled control cells to gemcitabine treatment. The apoptotic cell population increased in survivin-KD cells that were treated with gemcitabine in comparison to scrambled control cells, as observed by the cell cycle distribution and TUNEL assays. We found that survivin knockdown resulted in a reduction of glucose-regulated protein 78 (GRP78), which may be responsible for the observed increased survivin-KD cell sensitivity to gemcitabine. We conclude that survivin knockdown may contribute to a therapeutic effect of gemcitabine through GRP78 on HCC cells.

Retinoblastoma 94 Enhances Radiation Treatment of Esophageal Squamous Cell Carcinoma in Vitro and in Vivo

We performed the study to investigate whether adenovirus-mediated retinoblastoma 94 (RB94) gene transfer could enhance radiation treatment of esophageal squamous cell carcinoma (ESCC) in vitro and in vivo. ESCC cells (Kyse150 cell line) were cultivated in vitro and tumors originated from the cell line were propagated as xenografts in nude mice. Treatment with Ad-RB94 and/or ionizing radiation (IR) was carried out both in vitro and in vivo with Ad-LacZ control vector and blank control. Cell viability, cell cycle distribution, cell apoptosis, tumor growth and transfected gene expression were evaluated and tumor degeneration was analyzed. The data of quantification real-time PCR assays and immunohistochemistry staining using RB antibody indicated that RB94 was efficiently transfected into Kyse150 cells. In vitro, data of cell growth assay indicated that treatment with Ad-RB94 improved radiation treatment of Kyse150 cells. Tumor xenograft studies, pathological analysis of H.E. staining and Ki67 staining suggested transfecting RB94 enhanced tumor regression induced by radiation treatment in vivo. In addition, data of Annexin V, TUNEL and cell cycle distribution assays proposed combination treatment effectively induced cell apoptosis and cell cycle arresting in G2/M phase. In conclusion, transferring RB94 gene by the adenoviral vector enhances radiation treatment of ESCC.

Glucose-Regulated Protein 78 (GRP78) Mediated the Efficacy to Curcumin Treatment on Hepatocellular Carcinoma

Glucose-regulated protein 78 (GRP78) plays an important role in the therapeutic treatment and progression of cancer. However, little is known about the effect of GRP78 expression to curcumin in hepatocellular carcinoma (HCC). In this study, we generated GRP78 knockdown cells (GRP78KD) by a short interfering RNA (siRNA) technique. The antiproliferation effects of curcumin were determined by MTT assay, TUNEL assay, and cell cycle determination. We found that GRP78KD cells were more resistant to curcumin treatment compared with the parental cells in MTT assay. The apoptosis cell population was increased in scrambled-siRNA cells treated with curcumin compared with GRP78KD cells in cell cycle distribution and TUNEL assays. Finally, we found that knocking down GRP78 causes resistance to curcumin treatment through the suppression of caspase-3 and caspase-8 expression levels. We conclude that the expression level of GRP78 may contribute to the therapeutic effect of curcumin on HCC cells.

Effects of TRPM8 on the proliferation and motility of prostate cancer PC-3 cells

We investigated the effects of transient receptor potential M8 (TRPM8) channel on the proliferation and motility of androgen-independent prostate cancer PC-3 cells. After being permanently transfected with an empty vector and cDNA encoding the TRPM8 protein, cells were analysed for cell cycle distribution and motility using flow cytometry and scratch assay. Immunocytochemistry and Ca2+ imaging analysis revealed the overexpression of functional TRPM8 channel on both endoplasmic reticulum and plasma membrane of PC-3-TRPM8 cells. Cell cycle distribution and scratch assay analysis revealed that TRPM8 induced cell cycle arrest at the G0/G1 stage (P < 0.05) and facilitated the cell apoptosis induced by starvation (P < 0.05). Furthermore, TRPM8 inhibited the migration of PC-3-TRPM8 cells (P < 0.01) through the inactivation of focal-adhesion kinase. It appears that TRPM8 was not essential for the survival of PC-3 cells; however, the overexpression of TRPM8 had negative effects on the proliferation and migration of PC-3 cells. Thus, TRPM8 and its agonists may serve as important targets for the treatment of prostate cancer.

Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells

Puerariae radix (PR) is a popular natural herb and a traditional food in Asia, which has antithrombotic and anti-allergic properties and stimulates estrogenic activity. In the present study, we investigated the effects of the PR isoflavones puerarin, daidzein, and genistein on the growth of breast cancer cells. Our data revealed that after treatment with PR isoflavones, a dose-dependent inhibition of cell growth occurred in HS578T, MDA-MB-231, and MCF-7 cell lines. Results from cell cycle distribution and apoptosis assays revealed that PR isoflavones induced cell apoptosis through a caspase-3-dependent pathway and mediated cell cycle arrest in the G2/M phase. Furthermore, we observed that the serum metabolites of PR (daidzein sulfates/glucuronides) inhibited proliferation of the breast cancer cells at a 50% cell growth inhibition (GI 50) concentration of 2.35 μM. These results indicate that the daidzein constituent of PR can be metabolized to daidzein sulfates or daidzein glucuronides that exhibit anticancer activities. The protein expression levels of the active forms of caspase-9 and Bax in breast cancer cells were significantly increased by treatment with PR metabolites. These metabolites also increased the protein expression levels of p53 and p21. We therefore suggest that PR may act as a chemopreventive and/or chemotherapeutic agent against breast cancer by reducing cell viability and inducing apoptosis.